Polymeric carriers for immunohistochemistry and in situ hybridization

ABSTRACT

Certain disclosed embodiments of the present invention concern the synthesis, derivatization, conjugation to immunoglobulins and signal amplification based on discrete, relatively short polymers having plural reactive functional groups that react with plural molecules of interest. Reactive functional groups, such as hydrazides, may be derivatized with a variety of detectable labels, particularly haptens. The remaining reactive functional groups may be conjugated directly to a specific binding molecule, such as to the oxidized carbohydrate of the Fc region of the antibody. Disclosed conjugates display large signal amplification as compared to those based on molecules derivatized with single haptens, and are useful for assay methods, particularly multiplexed assays.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims the benefit of the earlier filing date ofU.S. Provisional Application No. 60/931,546, filed on May 23, 2007,which is incorporated herein by reference.

FIELD

The present invention concerns molecular conjugates, particularlyFc-specific conjugates, comprising a polymeric carrier having pluralreactive functional groups, embodiments of a method for making disclosedexemplary conjugates, and embodiments of a method for using theconjugates.

BACKGROUND

Biomolecular conjugates can be used in immunoassays for detectingspecific target molecules in a sample. Various conjugates are known,including antibody-detectable label conjugates and antibody-enzymeconjugates, and a number of methods have been developed for making theseconjugates. For example, antibody conjugates often are prepared usingcoupling reagents having at least two reactive groups. One of the groupsis used to couple to the antibody, and another functional group iscoupled to the detectable label. These coupling reactions can interferewith the performance of the conjugate for a desired purpose. Forexample, coupling can deactivate antibody-enzyme conjugates, such asthrough steric effects, deactivation of reactive functional groupscritical for appropriate functioning, changes in solubility, etc. As aresult, and despite prior efforts, there still is a need for molecularconjugates, and methods for their production and use, that providegreater assay sensitivity. Ventana Medical is the assignee of a numberof patents and applications in this general area, including: U.S. patentapplication Ser. No. 11/018,897, entitled Microwave Mediated Synthesisof Nucleic Acid Probes, published as U.S. publication No. 2005/0158770,on Jul. 21, 2005; U.S. Provisional Application No. 60/739,794, filed onNov. 23, 2005, and the corresponding utility application Ser. No.11/603,425, entitled “Molecular Conjugate;” U.S. Provisional ApplicationNo. 60/856,133, and the corresponding utility application Ser. No.11/982,627, entitled “Haptens, Hapten Conjugates, Compositions Thereofand Method for their Preparation and Use.” Each of these priorapplications is incorporated herein by reference. Example 12 of the '897patent discloses one method for making polyacrylamide hydrazide. The'897 patent application states that the “invention provides a method forpreparing a labeled cytosine, a labeled cytidine, or labeledcytidine-containing biomolecule, such as oligonucleotides, DNAmolecules, RNA molecules, proteins, peptides, or other biomolecules.”U.S. Patent Publication No. 2005/0158770, paragraph 0044. Moreover, theapplication states that “[l]inear polymers that are functionalized withfluorophores and nuclelophillic group(s) may also function as usefulreporter containing moities.” And “a preferred functionalized polymer ispolyacrylamide hydrazide functionalized with fluorophores, particularlyPAH of MW 10,000 to 20,000 bearing between 10 to 40 hydrazide groups perpolymer chain.” U.S. Patent Publication No. 2005/0158770, paragraph0068. Reporter groups are defined to include “any detectable moietycommonly used for labeling probes,” including haptens and proteins. U.S.Patent Publication No. 2005/0158770, paragraph 0053.

According to Scheme 11 of the '425 application, the Fc portion of anantibody is oxidized to form an aldehyde, and a thiolated hydrazide isthen coupled to the Fc portion of the antibody by reaction of ahydrazide nitrogen with the carbonyl. According to Scheme 13 of the '425application, a thiolated hydrazide coupled to the oxidized Fc portion ofan antibody is reacted with alkaline phosphatase having a thiol-reactivefunctional group to form a conjugate. And, according to Scheme 19, apolyacrylamide hydrazide is first synthesized, and then, as stated inExample 22:

-   -   In an appropriate solvent, the resulting PAH is reacted with a        thiolating agent, such as thiol-dPEG-NHS ester¹ (Quanta        Biodesign, Powell, Ohio) or Traut's reagent, to thiolate a        portion (for example, approximately 50-75%) of available        hydrazides (z=5 to 40) and provide a polymeric multifunctional        hydrazide thiol linker that can be used in the disclosed method.        ¹Applicants note that the reagent should be referred to as        S-acetyl-dPEG-NHS ester.        As presently understood, all embodiments of the PAH conjugates        disclosed in the '897 application have at least a portion of the        reactive hydrazide functional groups thiolated as disclosed in        Example 22. Moreover, it is the thiol group, provided by        thiolation of the hydrazide, that is the reactive moiety used to        form conjugates.

SUMMARY

Certain disclosed embodiments of the present invention concern thesynthesis, derivatization, conjugation to immunoglobulins and signalamplification based on discrete, relatively short polymers, e.g.,polyacrylamide hydrazide (PAH), having plural reactive functional groupsthat react with plural molecules of interest. Many of the disclosedembodiments concern substantially water soluble, or completely watersoluble, polymers. With reference to the exemplary PAH, such polymersare completely water soluble, and may display numerous reactivehydrazides, such as greater than zero hydrazide functional groups, andmore typically from about 5 up to at least 100 hydrazide groups.Reactive functional groups also can be quantified with reference to thepercentage of potential positions occupied by the pertinent functionalgroup, such as a hydrazide. For the present embodiments, the reactivefunctional group typically comprises at least 10%, and up to at least50%, of the possible positions that can be reactive functional groups.Reactive functional groups, such as the hydrazide, may be derivatizedwith a variety of haptens. The remaining hydrazides on the carrier maybe conjugated directly to the oxidized carbohydrate of the Fc region ofthe antibody. The low pKa of the hydrazide offers particular advantagein that the protonation of the aldehydes groups generated bycarbohydrate oxidation of the antibody facilitates the conjugation ofthe polymer hapten carrier. Moreover, the carrier is installed at the Fcregion of the antibody, away from the binding site of the IgG. Theresulting conjugate displays very large signal amplification as comparedto those based on Fc derivatized with single haptens.

One embodiment of the disclosed method concerns forming a molecularconjugate by coupling a specific binding molecule to a detectable labelthrough reactive hydrazide functional groups provided by apolyacrylamide hydrazide carrier. The polyacrylamide hydrazidepreferably is water soluble. The average molecular weight of thepolymeric portion can vary, and typically is from as low as 50 up to atleast about 100,000, more typically from about 1,000 to about 50,000,and even more typically from about 5,000 to about 40,000. Certaindisclosed embodiments use polyacrylamide hydrazides having an averagemolecular weight of about 10,000 or less. And, for particularembodiments, the hydrazide functional group of the polyacrylamidehydrazide is non-thiolated.

A particular disclosed embodiment comprises forming a first compound bycoupling the specific binding molecule to at least a portion of thereactive functional groups. At least a portion of remaining reactivefunctional groups of the first compound may then be coupled to adetectable label, such as a hapten. Alternatively, a polymeric carriercomprising plural reactive functional groups can be coupled to adetectable label, such as hapten. At least a portion of remainingreactive functional groups of the first compound may then coupled to aspecific binding molecule.

Particular embodiments concern antibodies as the specific bindingmolecule. For example, the polymeric carrier can be coupled to the Fcportion of the antibody through the reactive hydrazide functionalgroups. The antibody can be activated for reaction with the reactivefunctional groups, such as by chemically modifying a glycosylatedportion of the antibody. In certain working embodiments, the antibody ischemically activated by oxidation to form a carbonyl bearing compound,such as an aldehyde.

Many of the disclosed embodiments concern using haptens as a detectablelabel. The hapten can be any hapten now known or hereafter discovered ordeveloped that is suitable for practicing disclosed embodiments of themethod. Many haptens are known and frequently used for analyticalprocedures, such as di-nitrophenyl, biotin, digoxigenin, fluorescein,rhodamine, or combinations thereof. Other haptens have been specificallydeveloped by Ventana Medical Systems, including haptens selected fromoxazoles, pyrazoles, thiazoles, nitroaryls, benzofurans, triterpenes,ureas, thioureas, rotenoids, coumarins, cyclolignans, and combinationsthereof. Plural different haptens may be coupled to the polymericcarrier. Moreover, compounds, such as haptens, can be coupled to thepolymeric carrier using a linker, such as an NHS-PEG linker.

One specific disclosed embodiment concerns a method for forming amolecular conjugate, comprising coupling a specific binding molecule toa detectable label by a polyacrylamide hydrazide carrier. The carrierhas an average molecular weight of 10,000 or less and plural reactive,non-thiolated hydrazide functional groups.

A currently preferred embodiment using polyacrylamide hydrazide carrierscomprises providing a polyacrylamide polymeric carrier, typically havingan average molecular weight of about 10,000 or less, and comprisingplural reactive, non-thiolated hydrazide functional groups. The Fcportion of an antibody is oxidized to form an aldehyde. The polymericcarrier is coupled to the oxidized Fc portion of the antibody through atleast one reactive hydrazide functional group to form a first compound.The first compound is then coupled to at least one hapten through areactive hydrazide functional group to form a second compound.

The present invention also concerns conjugate comprising polyacrylamidehydrazides. For example, one disclosed conjugate embodiment comprises aspecific binding molecule covalently bonded to a detectable labelthrough a reactive hydrazide functional group provided by apolyacrylamide hydrazide linker. The conjugate also can comprise aPEG-based hydrazide linker, such as compounds having a functionalizedend and a distal end comprising a hydrazide or hydrazide derivativefunctional group. The detectable label typically is selected from anenzyme, a fluorophore, a luminophore, escent molecule, a hapten, afluorescent nanoparticle, or combinations thereof. Exemplary enzymesinclude alkaline phosphatase and horseradish peroxidase. Exemplary knownhaptens include di-nitrophenyl, biotin, digoxigenin, fluorescein,rhodamine, or combinations thereof. Additional exemplary haptens,developed by Ventana Medical, include oxazoles, pyrazoles, thiazoles,nitroaryls, benzofurans, triterpenes, ureas, thioureas, rotenoids,coumarins, cyclolignans, or combinations thereof. The specific bindingmolecule often is an antibody, including anti-hapten antibodies, andanti-antibody antibodies.

Polyacrylamide hydrazide carriers also can be used in a diagnostic assayprocess. One disclosed embodiment of such a process comprises contactinga sample with a specific binding molecule that binds specifically to atarget. The specific binding molecule is conjugated to a detectablelabel through the polyacrylamide hydrazide carrier. The specific bindingmolecule is then detected using the detectable label. The disclosedconjugates also can be used in a multiplexed assay. For example,disclosed embodiments include a multiplexed diagnostic assay for two ormore different targets in a sample, where the method comprisescontacting the sample with two or more specific binding molecules thatbind specifically to two or more different targets. The two or morespecific binding molecules are conjugated to different haptens through areactive hydrazide functional group of a polyacrylamide hapten carrier.The sample is then contacted with two or more different anti-haptenantibodies that can be detected separately.

While certain disclosed embodiments are directed particularly to usingpolyacrylamide hydrazide carriers, other polymeric carriers also arecontemplated. For these embodiments, one disclosed method for forming amolecular conjugate comprises coupling a specific binding molecule to adetectable label through reactive functional groups provided by apolymeric carrier. The polymeric carrier comprises a polymeric portionselected from polyacrylic acids, polyethyleneimines, polysaccharides,polyethylene-alt-maleic acids, polyamino acids or polyvinylpyrrolidones.The polymeric portion also includes plural reactive functional groupsselected from hydrazines, hydrazides, hydrazine derivatives, hydrazidederivatives, guanidines, aminoguanidines, hydroxyl amines, orcombinations thereof. Exemplary polysaccharide species may be selectedfrom carbohydrates, cellulose, carboxymethylcellulose, dextran,glycogen, polyhyaluronic acid and starch. Exemplary polyamino acids maybe selected from poly(arginine), poly(aspargine), poly(aspartic acid),poly(glutamic acid), poly(glutamine), poly(lysine) or combinationsthereof.

For particular embodiments, the method comprises forming a firstcompound by coupling the specific binding molecule to at least a portionof the reactive functional groups. At least a portion of any remainingnon-reacted functional groups of the first compound are coupled to adetectable label. Alternatively, the method may comprise forming a firstcompound by coupling a detectable label to at least a portion of thereactive functional groups, and then coupling at least a portion ofremaining reactive functional groups of the first compound to a specificbinding molecule.

One class of specific binding molecules is antibodies. The method maycomprise coupling the polymeric carrier to the Fc portion of theantibody through a reactive functional group. Again, the antibody may beactivated for reaction with the reactive functional groups, such as bychemically modifying a glycosylated portion of the antibody.

The specific binding molecule may be an antibody and the detectablelabel may be a hapten, as described with reference to the polyacrylamidehydrazide carriers. Plural different haptens may be coupled to thepolymeric carrier, and any one or more of such haptens may be coupled tothe carrier using a linker, such as PEG-based linker.

A particular embodiment of the method for forming a conjugate comprisesproviding a polymeric carrier comprising a polymeric portion selectedfrom polyacrylic acids, polyethyleneimines, polysaccharides,polyethylene-alt-maleic acids, polyamino acids or polyvinylpyrrolidones.The polymeric portion includes plural reactive functional groupsselected from hydrazines, hydrazides, hydrazine derivatives, hydrazidederivatives, guanidines, aminoguanidines, hydroxyl amines, orcombinations thereof. The Fc portion of an antibody is oxidized to forman aldehyde. The polymeric carrier is coupled to the oxidized Fc portionof the antibody and to at least one hapten, and potentially pluraldifferent haptens, through reactive functional groups.

One disclosed embodiment of the method comprises forming a molecularconjugate by providing a polymeric carrier comprising a polymericportion selected from polyacrylic acids, polyethyleneimines,polysaccharides, polyethylene-alt-maleic acids, polyamino acids orpolyvinylpyrrolidones. The polymeric portion also includes pluralreactive functional groups selected from hydrazines, hydrazides,hydrazine derivatives, hydrazide derivatives, guanidines,aminoguanidines, hydroxyl amines, or combinations thereof. A specificbinding molecule is coupled to the polymer carrier through a reactivefunctional group. A hapten also is coupled to the polymeric carrierthrough a reactive functional group, the hapten being selected fromoxazoles, pyrazoles, thiazoles, nitroaryls, benzofurans, triterpenes,ureas, thioureas, rotenoids, coumarins, cyclolignans, and combinationsthereof.

Molecular conjugates comprising polymeric carriers other thanpolyacrylamide hydrazides also are described. Exemplary conjugatescomprise a specific binding molecule coupled to a detectable labelthrough a polymeric carrier comprising a polymeric portion selected frompolyacrylamide-N-hydroxysuccinimide, polyacrylic acids,polyethyleneimines, polysaccharides, polyethylene-alt-maleic acids,polyamino acids or polyvinylpyrrolidones. Additional informationconcerning polyacrylamide-N-hydroxysuccinimide polymeric materials canbe found in Pollack et al., “Enzyme Immobilization by CondensationCopolymerization into Cross-Linked Polyacrylamide Gels,” JACS, Vol. 102,pages 6324-6336, which is incorporated herein by reference. The carrieralso includes plural reactive functional groups selected fromhydrazines, hydrazides, hydrazine derivatives, hydrazide derivatives,guanidines, aminoguanidines, hydroxyl amines, or combinations thereof.Exemplary polysaccharide species include carbohydrates, cellulose,carboxymethylcellulose, dextran, glycogen, polyhyaluronic acid andstarch. Certain embodiments also use oxidized species, particularlyoxidized polysaccharides. For example, dextran can be oxidized using asuitable oxidizing agent, including periodate or halogens, such asbromine, to produce a carbonyl-bearing species, typically an aldehyde,but potentially other carbonyl-bearing species, such as ketones.Exemplary polyamino acids include poly(arginine), poly(aspargine),poly(aspartic acid), poly(glutamic acid), poly(glutamine) andpoly(lysine). The carrier also can be a poly(guanidine) orpoly(aminoguanidine).

As with the polyacrylamide hydrazides, other disclosed polymericcarriers can be used for performing a diagnostic assay for a target in asample. Certain disclosed embodiments comprise contacting the samplewith a specific binding molecule that binds specifically to a target,wherein the specific binding molecule is conjugated to a detectablelabel through a polymeric carrier comprising a polymeric portionselected from polyacrylic acids, polyethyleneimines, polysaccharides,polyethylene-alt-maleic acids, polyamino acids, orpolyvinylpyrrolidones. The polymeric carrier includes plural reactivefunctional groups selected from hydrazines, hydrazides, hydrazinederivatives, hydrazide derivatives, guanidines, aminoguanidines,hydroxyl amines, or combinations thereof. The specific binding moleculeis detected using the detectable label. One embodiment of a multiplexeddiagnostic assay for two or more different targets in a sample comprisescontacting a sample with two or more specific binding molecules thatbind specifically to two or more different targets. The two or morespecific binding molecules are conjugated to different haptens through apolymeric hapten carrier having a polymeric portion selected frompolyacrylamide-N-hydroxysuccinimides, polyacrylic acids,polyethyleneimines, polysaccharides, polyethylene-alt-maleic acids,polyamino acids or polyvinylpyrrolidones. As with other disclosedembodiments, the polymeric carrier also includes plural reactivefunctional groups selected from hydrazines, hydrazides, hydrazinederivatives, hydrazide derivatives, guanidines, aminoguanidines,hydroxyl amines, or combinations thereof. The sample is then contactedwith two or more different anti-hapten antibodies that can be detectedseparately.

Comparisons of staining results also are provided, with single genedetection being established using the exemplary PAH disclosedembodiment. Similarly, the signal of streptavidin functionalized quantumdots is greatly enhanced in Fc derivatized polymeric biotin carrier atFc. Under in situ hybridization of the HER2 DNA gene probe, thepolymeric biotin carrier at Fc enhances the detection of quantum dotsignal compared to a non-polymeric biotin link. Certain disclosedembodiments concern one particular polymeric hapten carrier, namely PAH;however, many different carriers may be synthesized and used asdisclosed herein. These include, by way of example and withoutlimitation, polyacrylic, polyglucoside, polyglutamates, polylysines,polyaspartates suitably derivatized with the haptens may all be used.Similarly, the scope of the invention is not limited tobiotin-streptavidin-based systems. Rather, a wide variety of haptens andcorresponding antibodies conjugated to the signal generating entities,e.g. enzymes, nanoparticles, quantum dots, fluorophores,chemiluminophores, also may be used for the disclosed embodiments.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates one embodiment of a method for synthesizing anFc-specific haptenylated antibody conjugate.

FIG. 2 illustrates one embodiment of a method for synthesizing anFc-Specific Ab-hydrazide-functionalized polymer-polyhapten conjugate.

FIG. 3 is a photograph illustrating staining of Ki-67 on tonsil (655 nmfilter; 20× magnification) using a streptavidin-quantum dot 655conjugate with the Fc-specific biotinylated antibody.

FIG. 4 is a photograph illustrating staining of Ki-67 on tonsil (655 nmfilter; 20× magnification) using a streptavidin-quantum dot 655conjugate with the biotinylated polyacrylamide hydrazide antibody.

FIG. 5 is a photograph illustrating staining of a 1:10 dilution of Ki-67on tonsil (655 nm filter; 20× magnification) using astreptavidin-quantum dot 655 conjugate with the Fc-specific biotinylatedantibody.

FIG. 6 is a photograph illustrating staining of a 1:10 dilution of Ki-67on tonsil (Long Pass filter, Omega Optical XFO5-2; 20× magnification)using a streptavidin-quantum dot 655 conjugate with the biotinylatedpolyacrylamide hydrazide antibody.

FIG. 7 illustrates one embodiment of a staining protocol for QDot IHCdetection.

FIG. 8 is a photograph illustrating staining of tonsil tissue (Long Passfilter, Omega Optical XFO5-2; 20× magnification) using ananti-nitropyrazole-quantum dot 655 conjugate with an anti-lambdapolyacrylamide hydrazide nitropyrazole conjugate.

FIG. 9 is a photograph illustrating staining of tonsil tissue (Long Passfilter, Omega Optical XFO5-2; 20× magnification) using ananti-benzofurazan-quantum dot 585 conjugate with an anti-lambdapolyacrylamide hydrazide benzofurazan conjugate.

FIG. 10 is a photograph illustrating staining of tonsil (Long Passfilter, Omega Optical XFO5-2; 20× magnification) using ananti-dinitrophenyl quantum dot 605 conjugate with an anti-lambdapolyacrylamide hydrazide dinitrophenyl conjugate.

FIG. 11 is a photograph illustrating staining of tonsil tissue (LongPass filter, Omega Optical XFO5-2; 20× magnification) using ananti-thiazolesulfonamide-quantum dot 565 conjugate with an anti-lambdapolyacrylamide hydrazide thiazolesulfonamide conjugate.

FIG. 12 illustrates one embodiment of a staining protocol for HPV AP-ISHdetection.

FIG. 13 illustrates one embodiment of a staining protocol for HPV SISHdetection.

FIG. 14 is a photograph illustrating staining of HPV on xenographedtissue by silver detection (40× magnification) using an Fc-specificbiotinylated goat anti-rabbit antibody on CaSki cells (400-600 copies).

FIG. 15 is a photograph illustrating staining of HPV on xenographedtissue by silver detection (40× magnification) using an Fc-specificbiotinylated goat anti-rabbit antibody on HeLa cells (10-50 copies).

FIG. 16 is a photograph illustrating staining of HPV on xenographedtissue (40× magnification) biotinylated goat anti-rabbit antibody onSiHa cells (1-2 copies), Fc-specific biotinylated antibody.

FIG. 17 is a photograph illustrating staining of HPV on xenographedtissue by silver detection (40× magnification) using an Fc-specificbiotinylated goat anti-rabbit antibody on C33 cells (0 copies, negativecontrol).

FIG. 18 is a photograph illustrating staining of HPV on xenographedtissue by silver detection (40× magnification) using a polyacrylamidehydrazide biotinylated goat anti-rabbit antibody on CaSki cells (400-600copies).

FIG. 19 is a photograph illustrating staining of HPV on xenographedtissue by silver detection (40× magnification) using a polyacrylamidehydrazide biotinylated goat anti-rabbit antibody on HeLa cells (10-50copies).

FIG. 20 is a photograph illustrating staining of HPV on xenographedtissue by silver detection (40× magnification) using a polyacrylamidehydrazide biotinylated goat anti-rabbit antibody on SiHa cells (1-2copies).

FIG. 21 is a photograph illustrating staining of HPV on xenographedtissue by silver detection (40× magnification) using a polyacrylamidehydrazide biotinylated goat anti-rabbit antibody on C33 cells (0 copies,negative control).

FIG. 22 illustrates one embodiment of a staining protocol for multiplexIHC detection with quantum dots.

FIG. 23 is a photograph illustrating staining of tonsil tissue (LongPass filter, Omega Optical XFO5-2; 40× magnification) using ananti-nitropyrazole-quantum dot 655 conjugate with an anti-CD34polyacrylamide hydrazide nitropyrazole conjugate.

FIG. 24 is a photograph illustrating staining of tonsil tissue (LongPass filter, Omega Optical XFO5-2; 40× magnification) using ananti-benzofurazan-quantum dot 585 conjugate with an anti-Ki67polyacrylamide hydrazide benzofurazan conjugate.

FIG. 25 is a photograph illustrating staining of tonsil tissue (LongPass filter, Omega Optical XFO5-2; 40× magnification) using ananti-dinitrophenyl-quantum dot 605 conjugate with an anti-kappapolyacrylamide hydrazide dinitrophenyl conjugate.

FIG. 26 is a photograph illustrating staining of tonsil tissue (LongPass filter, Omega Optical XFO5-2; 40× magnification) using ananti-thiazolesulfonamide-quantum dot 565 conjugate with an anti-CD45polyacrylamide hydrazide thiazolesulfonamide conjugate.

FIG. 27 is a photograph illustrating staining of tonsil tissue (OlympusDP71; UPlanSApo; 40× magnification) using an anti-dinitrophenyl-HRPconjugate/DAB with an anti-kappa polyvinylpyrrolidone hydrazidedinitrophenyl conjugate.

FIG. 28 is a photograph illustrating staining of tonsil tissue (LongPass filter, Omega Optical XFO5-2; 40× magnification) using ananti-dinitrophenyl-quantum dot 655 conjugate with an anti-kappapolyvinylpyrrolidone hydrazide dinitrophenyl conjugate.

FIG. 29 is a photograph illustrating staining of tonsil tissue (OlympusDP71; UPlanSApo; 40× magnification) using an anti-dinitrophenyl-HRPconjugate/DAB with an anti-kappa polyisobutylene-co-maleic hydrazidedinitrophenyl conjugate.

FIG. 30 is a photograph illustrating staining of tonsil tissue (LongPass filter, Omega Optical XFO5-2; 40× magnification) using ananti-dinitrophenyl-quantum dot 655 conjugate with an anti-kappapolyisobutylene-co-maleic hydrazide dinitrophenyl conjugate.

FIG. 31 is a photograph illustrating staining of tonsil tissue (OlympusDP71; UPlanSApo; 40× magnification) using an anti-dinitrophenyl-HRPconjugate/DAB with an anti-kappa polyacrylic acid hydrazidedinitrophenyl conjugate.

FIG. 32 is a photograph illustrating staining of tonsil tissue (LongPass filter, Omega Optical XFO5-2; 40× magnification) using ananti-dinitrophenyl-quantum dot 655 conjugate with an anti-kappapolyacrylic acid hydrazide dinitrophenyl conjugate.

DETAILED DESCRIPTION I. Abbreviations

-   -   Ab—antibody    -   (Ab-AP)—antibody-alkaline phosphatase conjugate    -   ABS—acetate buffered saline.    -   AP—alkaline phosphatase    -   BSA—bovine serum albumin    -   CMV—cytomegalovirus    -   dPEG—discrete polyethylene glycol, such as dPEG₄, which refers        to a discretely sized PEG compound having 4 ether oxygen atoms.    -   EBER—Epstein-Barr virus early RNA    -   DL—detectable label    -   Fc—fragment crystallizable    -   HRP—horseradish peroxidase    -   IHC—immunohistochemistry    -   ISH—in situ hybridization    -   MAL—maleimide    -   MBCH—mercaptobutyric acid carbohydrazide    -   MBH—mercaptobutyric acid hydrazide    -   NHS—N-hydroxy-succinimide    -   SBM—specific binding molecule    -   SEC—size exclusion chromatography    -   SISH—silver in situ hybridization

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Publishers, 1994 (ISBN0632021829); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by Wiley, John& Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. Also, as used herein, the term “comprises” means“includes.” Hence “comprising A or B” means including A, B, or A and B.It is further to be understood that all amino acid sizes, and allmolecular weight or molecular mass values, given for polypeptides orother compounds are approximate, and are provided for description.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

In order to facilitate review of the various examples of thisdisclosure, the following explanations of specific terms are provided:

Amplification: Certain embodiments of the present invention allow asingle target to be detected using plural visualization complexes, wherethe complexes can be the same or different, to facilitate identificationand/or quantification of a particular target.

Analog, Derivative or Mimetic: An analog is a molecule that differs inchemical structure from a parent compound, for example a homolog(differing by an increment in the chemical structure, such as adifference in the length of an alkyl chain), a molecular fragment, astructure that differs by one or more functional groups, a change inionization. Structural analogs are often found using quantitativestructure activity relationships (QSAR), with techniques such as thosedisclosed in Remington (The Science and Practice of Pharmacology, 19thEdition (1995), chapter 28). A derivative is a biologically activemolecule derived from the base structure. A mimetic is a molecule thatmimics the activity of another molecule, such as a biologically activemolecule. Biologically active molecules can include chemical structuresthat mimic the biological activities of a compound.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects, for example, humans, non-human primates,dogs, cats, horses, and cows.

Antibody: “Antibody” collectively refers to immunoglobulins orimmunoglobulin-like molecules (including by way of example and withoutlimitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, andsimilar molecules produced during an immune response in any vertebrate,for example, in mammals such as humans, goats, rabbits and mice), aswell as non-mammalian species, such as shark immunoglobulins. “Antibody”also includes antibody fragments that specifically bind to a molecule ofinterest (or a group of highly similar molecules of interest) to thesubstantial exclusion of binding to other molecules (for example,antibodies and antibody fragments that have a binding constant for themolecule of interest that is at least 10³ M⁻¹ greater, at least 10⁴ M⁻¹greater or at least 10⁵ M⁻¹ greater than a binding constant for othermolecules in a biological sample.

More particularly, “antibody” refers to a polypeptide ligand comprisingat least a light chain or heavy chain immunoglobulin variable regionwhich specifically recognizes and binds an epitope of an antigen.Antibodies are composed of a heavy and a light chain, each of which hasa variable region, termed the variable heavy (V_(H)) region and thevariable light (V_(L)) region. Together, the V_(H) region and the V_(L)region are responsible for binding the antigen recognized by theantibody.

This includes intact immunoglobulins and the variants and portions ofthem well known in the art. Antibody fragments include proteolyticantibody fragments [such as F(ab′)₂ fragments, Fab′ fragments, Fab′-SHfragments and Fab fragments as are known in the art], recombinantantibody fragments (such as sFv fragments, dsFv fragments, bispecificsFv fragments, bispecific dsFv fragments, F(ab)′₂ fragments, singlechain Fv proteins (“scFv”), disulfide stabilized Fv proteins (“dsFv”),diabodies, and triabodies (as are known in the art), and camelidantibodies (see, for example, U.S. Pat. Nos. 6,015,695; 6,005,079-5,874,541; 5,840,526; 5,800,988; and 5,759,808). An scFv protein is afusion protein in which a light chain variable region of animmunoglobulin and a heavy chain variable region of an immunoglobulinare bound by a linker, while in dsFvs, the chains have been mutated tointroduce a disulfide bond to stabilize the association of the chains.The term also includes genetically engineered forms such as chimericantibodies (for example, humanized murine antibodies), heteroconjugateantibodies (such as, bispecific antibodies). See also, Pierce Catalogand Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J.,Immunology, 3^(rd) Ed., W.H. Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (□) and kappa (k). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs”. The extent of theframework region and CDRs have been defined (see, Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991, which is hereby incorporated byreference). The Kabat database is now maintained online. The sequencesof the framework regions of different light or heavy chains arerelatively conserved within a species. The framework region of anantibody, that is the combined framework regions of the constituentlight and heavy chains, serves to position and align the CDRs inthree-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. An antibody that binds RET will have a specificV_(H) region and the V_(L) region sequence, and thus specific CDRsequences. Antibodies with different specificities (i.e. differentcombining sites for different antigens) have different CDRs. Although itis the CDRs that vary from antibody to antibody, only a limited numberof amino acid positions within the CDRs are directly involved in antigenbinding. These positions within the CDRs are called specificitydetermining residues (SDRs).

Antigen: A compound, composition, or substance that may be specificallybound by the products of specific humoral or cellular immunity, such asan antibody molecule or T-cell receptor. Antigens can be any type ofmolecule including, for example, haptens, simple intermediarymetabolites, sugars (e.g., oligosaccharides), lipids, and hormones aswell as macromolecules such as complex carbohydrates (e.g.,polysaccharides), phospholipids, and proteins. Common categories ofantigens include, but are not limited to, viral antigens, bacterialantigens, fungal antigens, protozoa and other parasitic antigens, tumorantigens, antigens involved in autoimmune disease, allergy and graftrejection, toxins, and other miscellaneous antigens. In one example, anantigen is a Bacillus antigen, such as γPGA.

Avidin: Any type of protein that specifically binds biotin to thesubstantial exclusion of other small molecules that might be present ina biological sample. Examples of avidin include avidins that arenaturally present in egg white, oilseed protein (e.g., soybean meal),and grain (e.g., corn/maize) and streptavidin, which is a protein ofbacterial origin.

Binding Affinity: The tendency of one molecule to bind (typicallynon-covalently) with another molecule, such as the tendency of a memberof a specific binding pair for another member of a specific bindingpair. A binding affinity can be measured as a binding constant, whichbinding affinity for a specific binding pair (such as anantibody/antigen pair) can be at least 1×10⁵ M⁻¹, such as at least 1×10⁶M⁻¹, at least 1×10⁷ M⁻¹ or at least 1×108 M⁻¹. In one embodiment,binding affinity is calculated by a modification of the Scatchard methoddescribed by Frankel et al., Mol. Immunol., 16:101-106, 1979. In anotherembodiment, binding affinity is measured by an antigen/antibodydissociation rate. In yet another embodiment, a high binding affinity ismeasured by a competition radioimmunoassay. In several examples, a highbinding affinity for an antibody/antigen pair is at least about 1×10⁸M⁻¹. In other embodiments, a high binding affinity is at least about1.5×10⁸ M⁻¹, at least about 2.0×10⁸ M⁻¹, at least about 2.5×10⁸ M⁻¹, atleast about 3.0×10⁸ M⁻¹, at least about 3.5×10⁸ M⁻¹, at least about4.0×10⁸ M⁻¹, at least about 4.5×10⁸ M⁻¹, or at least about 5.0×10⁸ M⁻¹.

Carrier: A molecule to which a hapten or an antigen can be bound.Carrier molecules include immunogenic carriers and specific-bindingcarriers. When bound to an immunogenic carrier, the bound molecule maybecome immunogenic. Immunogenic carriers may be chosen to increase theimmunogenicity of the bound molecule and/or to elicit antibodies againstthe carrier, which are diagnostically, analytically, and/ortherapeutically beneficial. Covalent linking of a molecule to a carriercan confer enhanced immunogenicity and T-cell dependence (Pozsgay etal., PNAS 96:5194-97, 1999; Lee et al., J. Immunol. 116:1711-18, 1976;Dintzis et al., PNAS 73:3671-75, 1976). Useful carriers includepolymeric carriers, which can be natural (for example, proteins frombacteria or viruses), semi-synthetic or synthetic materials containingone or more functional groups to which a reactant moiety can beattached. Specific binding carriers can by any type of specific bindingmoiety, including an antibody, an avidin, etc.

Chimeric Antibody: An antibody that has framework residues from onespecies, such as human, and CDRs (which generally confer antigenbinding) from another species, such as a murine antibody thatspecifically binds RET.

Conjugate: A “conjugate” refers to two or more molecules (and/ormaterials such as nanoparticles) that are covalently linked into alarger construct. In some embodiments, a conjugate includes one or morebiomolecules (such as peptides, proteins, enzymes, sugars,polysaccharides, lipids, glycoproteins, and lipoproteins) covalentlylinked to one or more other molecules, such as one or more otherbiomolecules. In other embodiments, a conjugate includes one or morespecific-binding molecules (such as antibodies) covalently linked to oneor more detectable labels (such as a fluorophore, a luminophore,fluorescent nanoparticles, haptens, enzymes and combinations thereof).

Conjugating, Coupling, Joining, Bonding or Linking: Covalently linkingone molecule to another molecule to make a larger molecule. For example,making two polypeptides into one contiguous polypeptide molecule, or tocovalently attaching a hapten or other molecule to a polypeptide, suchas an scFv antibody. In the specific context, the terms includereference to joining a ligand, such as an antibody moiety, to aneffector molecule (“EM”). The linkage can be either by chemical orrecombinant means.

Coupling a specific binding molecule to a detectable label throughreactive hydrazide functional groups: Refers to covalently linking aspecific binding molecule to another molecule by a direct covalent bondto a nitrogen atom of a hydrazide functional group.

Detectable Label: A molecule or material that can produce a detectable(such as visually, electronically or otherwise) signal that indicatesthe presence and/or concentration of the label in a sample. Whenconjugated to a specific binding molecule, the detectable label can beused to locate and/or quantify the target to which the specific bindingmolecule is directed. Thereby, the presence and/or concentration of thetarget in a sample can be detected by detecting the signal produced bythe detectable label. A detectable label can be detected directly orindirectly, and several different detectable labels conjugated todifferent specific-binding molecules can be used in combination todetect one or more targets. For example, a first detectable label suchas a hapten conjugated to an antibody specific to a target can bedetected indirectly through the use of a second detectable label that isconjugated to a molecule that specifically binds the first detectablelabel. Multiple detectable labels that can be separately detected can beconjugated to different specific binding molecules that specificallybind different targets to provide a multiplexed assay that can providesimultaneous detection of the multiple targets in a sample. A detectablesignal can be generated by any known or yet to be discovered mechanismincluding absorption, emission and/or scattering of a photon (includingradio frequency, microwave frequency, infrared frequency, visiblefrequency and ultra-violet frequency photons). Detectable labels includecolored, fluorescent, phosphorescent and luminescent molecules andmaterials, catalysts (such as enzymes) that convert one substance intoanother substance to provide a detectable difference (such as byconverting a colorless substance into a colored substance or vice versa,or by producing a precipitate or increasing sample turbidity), haptensthat can be detected through antibody-hapten binding interactions usingadditional detectably labeled antibody conjugates, and paramagnetic andmagnetic molecules or materials. Particular examples of detectablelabels include enzymes such as horseradish peroxidase, alkalinephosphatase, acid phosphatase, glucose oxidase, β-galactosidase orβ-glucuronidase; fluorphores such as fluoresceins, luminophores,coumarins, BODIPY dyes, resorufins, and rhodamines (many additionalexamples of fluorescent molecules can be found in The Handbook—A Guideto Fluorescent Probes and Labeling Technologies, Molecular Probes,Eugene, Oreg.); nanoparticles such as quantum dots (obtained, forexample, from QuantumDot Corp, Invitrogen Nanocrystal Technologies,Hayward, Calif.; see also, U.S. Pat. Nos. 6,815,064, 6,682,596 and6,649,138, each of which patents is incorporated by reference herein);metal chelates such as DOTA and DPTA chelates of radioactive orparamagnetic metal ions like Gd³⁺; and liposomes, for example, liposomescontaining trapped fluorescent molecules. Where the detectable labelincludes an enzyme, a detectable substrate such as a chromogen, afluorogenic compound, or a luminogenic compound can be used incombination with the enzyme to generate a detectable signal (A widevariety of such compounds are commercially available, for example, fromInvitrogen Corporation, Eugene Oreg.). Particular examples ofchromogenic compounds include diaminobenzidine (DAB),4-nitrophenylphospate (pNPP), fast red, bromochloroindolyl phosphate(BCIP), nitro blue tetrazolium (NBT), BCIP/NBT, fast red, AP Orange, APblue, tetramethylbenzidine (TMB), 2,2′-azino-di-[3-ethylbenzothiazolinesulphonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN),nitrophenyl-β-D-galactopyranoside (ONPG), o-phenylenediamine (OPD),5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal),methylumbelliferyl-β-D-galactopyranoside (MU-Gal),p-nitrophenyl-α-D-galactopyranoside (PNP),5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazol (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blueand tetrazolium violet. Alternatively, an enzyme can be used in ametallographic detection scheme. Metallographic detection methodsinclude using an enzyme such as alkaline phosphatase in combination witha water-soluble metal ion and a redox-inactive substrate of the enzyme.The substrate is converted to a redox-active agent by the enzyme, andthe redox-active agent reduces the metal ion, causing it to form adetectable precipitate. (See, for example, co-pending U.S. patentapplication Ser. No. 11/015,646, filed Dec. 20, 2004, PCT PublicationNo. 2005/003777 and U.S. Patent Application Publication No.2004/0265922; each of which is incorporated by reference herein).Metallographic detection methods include using an oxido-reductase enzyme(such as horseradish peroxidase) along with a water soluble metal ion,an oxidizing agent and a reducing agent, again to for form a detectableprecipitate. (See, for example, U.S. Pat. No. 6,670,113, which isincorporated by reference herein). Haptens are small molecules that arespecifically bound by antibodies, although by themselves they will notelicit an immune response in an animal and must first be attached to alarger carrier molecule such as a protein to generate an immuneresponse. Examples of haptens include di-nitrophenyl, biotin,digoxigenin, and fluorescein. Additional examples of oxazole, pyrazole,thiazole, nitroaryl, benzofuran, triperpene, urea, thiourea, rotenoid,coumarin and cyclolignan haptens are disclosed in co-pending U.S.Provisional Patent Application No., 60/856,133, filed Nov. 1, 2006,which is incorporated by reference herein.

Epitope: An antigenic determinant. These are particular chemical groupsor contiguous or non-contiguous peptide sequences on a molecule that areantigenic, that is, that elicit a specific immune response. An antibodybinds a particular antigenic epitope.

Fc-specific Conjugate: A conjugate of an immunoglobulin (or fragmentthereof) in which a second molecule (such as a detectable label) iscovalently bonded to the glycosylated portion of the immunoglobulin (ora fragment of an immunoglobulin that retains the glycosylated portion).The glycosylated portion of an immunoglobulin is found in the Fc-region,which is a region of an immunoglobulin that is located on the heavychains of the immunoglobulin at positions outside of the portion of theimmunoglobulin that is responsible for the specific binding activity ofthe immunoglobulin.

Hapten: A molecule, typically a small molecule that can combinespecifically with an antibody, but typically is substantially incapableof being immunogenic except in combination with a carrier molecule.

Homopolymer: This term refers to a polymer formed by the bondingtogether of multiple units of a single type of molecular species, suchas a single monomer (for example, an amino acid).

Humanized Antibody: An antibody comprising a humanized light chain and ahumanized heavy chain immunoglobulin. A humanized antibody binds to thesame antigen as the donor antibody that provides the CDRs. The acceptorframework of a humanized immunoglobulin or antibody may have a limitednumber of substitutions by amino acids taken from the donor framework.Humanized or other monoclonal antibodies can have additionalconservative amino acid substitutions which have substantially no effecton antigen binding or other immunoglobulin functions. Humanizedimmunoglobulins can be constructed by means of genetic engineering (seefor example, U.S. Pat. No. 5,585,089).

Humanized Immunoglobulin: an immunoglobulin including a human frameworkregion and one or more CDRs from a non-human (for example a mouse, rat,or synthetic) immunoglobulin. The non-human immunoglobulin providing theCDRs is termed a “donor,” and the human immunoglobulin providing theframework is termed an “acceptor.” In one embodiment, all the CDRs arefrom the donor immunoglobulin in a humanized immunoglobulin. Constantregions need not be present, but if they are, they must be substantiallyidentical to human immunoglobulin constant regions, i.e., at least about85-90%, such as about 95% or more identical. Hence, all parts of ahumanized immunoglobulin, except possibly the CDRs, are substantiallyidentical to corresponding parts of natural human immunoglobulinsequences.

Hydrazide or Hydrazide Group: A hydrazide group (—CO—NH—NH₂); acarbohydrazide group (—NH—NH—CO—NH—NH₂); a semicarbazide group(—NH—CO—NH—NH₂); a thiosemicarbazide group (—NH—CS—NH—NH₂); athiocarbazide group (—NH—NH—CS—NH—NH₂); a carbonic acid dihydrazinegroup (—NH—CO—NH—NH—CO—NH—NH₂) or a sulfur containing derivativethereof; or a hydrazine carboxylate group (—O—CO—NH—NH₂) or asulfur-containing derivative thereof.

Hydrazide-reactive Group: A group of atoms that can react with and forma covalent bond to a hydrazide group. Aldehyde and ketone groups areexamples of hydrazide-reactive groups. Hydrazide-reactive groups can bean intrinsic part of a molecule or can be introduced to a molecule. Onemethod for introducing an aldehyde group (a hydrazide-reactive group)into polysaccharides and glycoproteins (including antibodies) is byoxidation such as periodate-mediated oxidation of vicinal diols. Inaddition, double bonds in unsaturated fatty acids and ceramides can beconverted to diols by osmium tetroxide and then oxidized by periodate toaldehydes. Furthermore, N-terminal serine and threonine residues ofpeptides and proteins can be selectively oxidized by periodate toaldehyde groups, permitting selective modification of certain proteinssuch as corticotrophin and β-lactamase. Modification ofperiodate-oxidized antibodies does not typically inactivate theantibody. Varying the concentration of sodium periodate during theoxidation reaction gives some specificity with regard to the types ofsugar residues that are modified. For example, sodium periodate at aconcentration of 1 mM at 0° C. typically cleaves only at the adjacenthydroxyls between carbon atoms 7, 8 and 9 of sialic acid residues.Oxidizing polysaccharides using 10 mM or greater concentrations ofsodium periodate results in oxidation of sugar residues other thansialic acid, thereby creating many aldehydes on a given polysaccharide.A suitable general protocol is described by Hermanson, “BioconjugateTechniques,” Academic Press, San Diego, 1996, ISBN 0-12-342336-8, whichis incorporated by reference herein. Another method for introducingaldehydes into biomolecules is through the use of specific sugaroxidases, for example, galactose oxidase, which is an enzyme thatoxidizes terminal galactose residues to aldehydes, particularly inglycoproteins. When galactose residues are penultimate to sialic acidresidues, neuramidase can be used to remove the sialic acid residue andexpose galactose as the terminal residue. A protocol for using acombination of neuramidase and galactose oxidase to oxidize galactoseresidues to provide a reactive aldehyde group is provided in Hermanson,“Bioconjugate Techniques,” Academic Press, San Diego, 1996, ISBN0-12-342336-8, which is incorporated by reference herein. Aldehydes alsocan be introduced to a molecule by reacting an amine group of a moleculewith an NHS-aldehyde such as succinimidyl p-formylbenzoate (SFB) orsuccinimidyl p-formylphenoxyacetate (SFPA) (Invitrogen Corp., Eugene,Oreg.). Alternatively, bis-aldehyde compounds such as glutaraldehyde canbe used to modify an amine group to provide an aldehyde group. Again,suitable protocols are provided in Hermanson, “Bioconjugate Techniques,”Academic Press, San Diego, 1996, ISBN 0-12-342336-8, which isincorporated by reference herein.

Hydrazine, Hydrazine Derivatives: Chemical compounds or moietiestypically having a formula N₂H₄. Hydrazine derivatives are compounds ormoieties where at least one, and potentially plural hydrogen atoms ofhydrazine are replaced with other groups, such as aliphatic groups,particularly alkyl groups, and even more typically lower alkyl groups.

Immune Response: A response of a cell of the immune system, such as aB-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus. Animmune response can include any cell of the body involved in a hostdefense response for example, an epithelial cell that secretesinterferon or a cytokine. An immune response includes, but is notlimited to, an innate immune response or inflammation.

Immunogenic Conjugate or Composition: A term used herein to mean acomposition useful for stimulating or eliciting a specific immuneresponse (or immunogenic response) in a vertebrate. In some embodiments,the immunogenic response is protective or provides protective immunity,in that it enables the vertebrate animal to better resist infection ordisease progression from the organism against which the immunogeniccomposition is directed. One specific example of a type of immunogeniccomposition is a vaccine.

Immunogen: A compound, composition, or substance which is capable, underappropriate conditions, of stimulating the production of antibodies or aT-cell response in an animal, including compositions that are injectedor absorbed into an animal.

Immunologically Effective Dose: An immunologically effective dose of thedisclosed conjugates of the disclosure is therapeutically effective andwill prevent, treat, lessen, or attenuate the severity, extent orduration of a disease or condition.

Immunologically Reactive Conditions: Includes reference to conditionswhich allow an antibody raised against a particular epitope to bind tothat epitope to a detectably greater degree than, and/or to thesubstantial exclusion of, binding to substantially all other epitopes.Immunologically reactive conditions are dependent upon the format of theantibody binding reaction and typically are those utilized inimmunoassay protocols or those conditions encountered in vivo. SeeHarlow & Lane, supra, for a description of immunoassay formats andconditions. The immunologically reactive conditions employed in themethods are “physiological conditions” which include reference toconditions (such as temperature, osmolarity, pH) that are typical insidea living mammal or a mammalian cell. While it is recognized that someorgans are subject to extreme conditions, the intra-organismal andintracellular environment normally lies around pH 7 (i.e., from pH 6.0to pH 8.0, more typically pH 6.5 to 7.5), contains water as thepredominant solvent, and exists at a temperature above 0° C. and below50° C. Osmolarity is within the range that is supportive of cellviability and proliferation.

Isolated: An “isolated” microorganism (such as a virus, bacterium,fungus, or protozoan) has been substantially separated or purified awayfrom microorganisms of different types, strains, or species.Microorganisms can be isolated by a variety of techniques, includingserial dilution and culturing.

An “isolated” biological component (such as a protein or organelle) hasbeen substantially separated or purified away from other biologicalcomponents in the cell of the organism in which the component naturallyoccurs, such as other chromosomal and extra-chromosomal DNA and RNA,proteins, and organelles. Proteins that have been “isolated” includeproteins purified by standard purification methods. The term alsoembraces proteins prepared by recombinant expression in a host cell, aswell as chemically synthesized proteins, or fragments thereof.

Ki-67: a nuclear antigen (protein) involved in cellular proliferationuseful for cancer diagnosis as it is expressed in all stages of the cellcycle except for G0 (the resting phase).

Linker Peptide: A peptide within an antibody binding fragment (such asan Fv fragment) which serves to indirectly bond the variable heavy chainto the variable light chain. “Linker” can also refer to a peptideserving to link a targeting moiety, such as a scFv, to an effectormolecule, such as a cytotoxin or a detectable label.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Molecule of Interest or Target: A molecule for which the presence,location and/or concentration is to be determined. Examples of moleculesof interest include proteins tagged with haptens.

Monoclonal Antibody: An antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

Multiplex, -ed, -ing: Embodiments of the present invention allowmultiple targets in a sample to be detected substantiallysimultaneously, or sequentially, as desired, using plural differentconjugates. Multiplexing can include identifying and/or quantifyingpeptides, proteins, both individually and in any and all combinations.Multiplexing also can include detecting two or more of a messenger and aprotein in a cell in its anatomic context.

Nanoparticle: A nanoscale particle with a size that is measured innanometers, for example, a nanoscopic particle that has at least onedimension of less than about 100 nm. Examples of nanoparticles includeparamagnetic nanoparticles, superparamagnetic nanoparticles, metalnanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers(such as with covalently attached metal chelates), nanofibers,nanohorns, nano-onions, nanorods, nanoropes and quantum dots. Ananoparticle can produce a detectable signal, for example, throughabsorption and/or emission of photons (including radio frequency andvisible photons) and plasmon resonance.

Neoplasia and Tumor: The process of abnormal and uncontrolled growth ofcells. Neoplasia is one example of a proliferative disorder.

The product of neoplasia is a neoplasm (a tumor), which is an abnormalgrowth of tissue that results from excessive cell division. A tumor thatdoes not metastasize is referred to as “benign.” A tumor that invadesthe surrounding tissue and/or can metastasize is referred to as“malignant.” Examples of hematological tumors include leukemias,including acute leukemias (such as acute lymphocytic leukemia, acutemyelocytic leukemia, acute myelogenous leukemia and myeloblastic,promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronicleukemias (such as chronic myelocytic (granulocytic) leukemia, chronicmyelogenous leukemia, and chronic lymphocytic leukemia), polycythemiavera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent andhigh grade forms), multiple myeloma, Waldenstrom's macroglobulinemia,heavy chain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostatecancer, hepatocellular carcinoma, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroidcarcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervicalcancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors(such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma).

Non-thiolated: Conjugates that do not include a sulfur atom between thepolymeric backbone of a carrier and the specific binding molecule.

Peptide Nucleic Acid: Peptide nucleic acids are nucleic acid mimicscomprising a psuedopeptide backbone. Peptide nucleic acid oligomers formstable duplex structures with complementary DNA, RNA (or PNA) oligomers,and they can also bind to targets in duplex DNA by helix invasion. Thehistory, properties and applications of peptide nucleic acids in drugdiscovery and DNA detection are presented in the book “Peptide NucleicAcids.” Peptide nucleic acids were originally designed as ligands forrecognizing double stranded DNA. The nucleobases of DNA were retained,but the deoxyribose phosphodiester backbone of DNA was replaced by apseudo-peptide backbone. Exemplary peptide nucleic acids includehomo-thymine peptide nucleic acids.

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used. The terms “polypeptide” or “protein” as used herein areintended to encompass any amino acid sequence and include modifiedsequences such as glycoproteins. The term “polypeptide” is specificallyintended to cover naturally occurring proteins, as well as those whichare recombinantly or synthetically produced.

The term “residue” or “amino acid residue” includes reference to anamino acid that is incorporated into a protein, polypeptide, or peptide.

Protein: A molecule, particularly a polypeptide, comprised of aminoacids.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purifiedpeptide, protein, conjugate, or other active compound is one that isisolated in whole or in part from proteins or other contaminants.Generally, substantially purified peptides, proteins, conjugates, orother active compounds for use within the disclosure comprise more than80% of all macromolecular species present in a preparation prior toadmixture or formulation of the peptide, protein, conjugate or otheractive compound with a pharmaceutical carrier, excipient, buffer,absorption enhancing agent, stabilizer, preservative, adjuvant or otherco-ingredient in a complete pharmaceutical formulation for therapeuticadministration. More typically, the peptide, protein, conjugate or otheractive compound is purified to represent greater than 90%, often greaterthan 95% of all macromolecular species present in a purified preparationprior to admixture with other formulation ingredients. In other cases,the purified preparation may be essentially homogeneous, wherein othermacromolecular species are not detectable by conventional techniques.

Quantum Dot: A nanoscale particle that exhibits size-dependentelectronic and optical properties due to quantum confinement. Quantumdots have, for example, been constructed of semiconductor materials(e.g., cadmium selenide and lead sulfide) and from crystallites (grownvia molecular beam epitaxy), etc. A variety of quantum dots havingvarious surface chemistries and fluorescence characteristics arecommercially available from Invitrogen Corporation, Eugene, Oreg. (see,for example, U.S. Pat. Nos. 6,815,064, 6,682,596 and 6,649,138, each ofwhich patents is incorporated by reference herein). Quantum dots arealso commercially available from Evident Technologies (Troy, N.Y.).Other quantum dots include alloy quantum dots such as ZnSSe, ZnSeTe,ZnSTe, CdSSe, CdSeTe, ScSTe, HgSSe, HgSeTe, HgSTe, ZnCdS, ZnCdSe,ZnCdTe, ZnHgS, ZnHgSe, ZnHgTe, CdHgS, CdHgSe, CdHgTe, ZnCdSSe, ZnHgSSe,ZnCdSeTe, ZnHgSeTe, CdHgSSe, CdHgSeTe, InGaAs, GaAlAs, and InGaN quantumdots (Alloy quantum dots and methods for making the same are disclosed,for example, in US Application Publication No. 2005/0012182 and PCTPublication WO 2005/001889).

Reactive Groups: Formulas throughout this application refer to “reactivegroups,” which can be any of a variety of groups suitable for coupling afirst unit to a second unit as described herein. For example, thereactive group might be an amine-reactive group, such as anisothiocyanate, an isocyanate, an acyl azide, an NHS ester, an acidchloride, such as sulfonyl chloride, aldehydes and glyoxals, epoxidesand oxiranes, carbonates, arylating agents, imidoesters, carbodiimides,anhydrides, and combinations thereof. Suitable thiol-reactive functionalgroups include haloacetyl and alkyl halides, maleimides, aziridines,acryloyl derivatives, arylating agents, thiol-disulfide exchangereagents, such as pyridyl disulfides, TNB-thiol, and disulfidereductants, and combinations thereof. Suitable carboxylate-reactivefunctional groups include diazoalkanes, diazoacetyl compounds,carbonyldiimidazole compounds, and carbondiimides. Suitablehydroxyl-reactive functional groups include epoxides and oxiranes,carbonyldiimidazole, N,N′-disuccinimidyl carbonates orN-hydroxysuccinimidyl chloroformates, periodate oxidizing compounds,enzymatic oxidation, alkyl halogens, and isocyanates. Aldehyde andketone-reactive functional groups include hydrazines, Schiff bases,reductive amination products, Mannich condensation products, andcombinations thereof. Active hydrogen-reactive compounds includediazonium derivatives, mannich condensation products, iodinationreaction products, and combinations thereof. Photoreactive chemicalfunctional groups include aryl azides, halogenated aryl azides,benzophonones, diazo compounds, diazirine derivatives, and combinationsthereof.

Sample: The term “sample” refers to any liquid, semi-solid or solidsubstance (or material) in or on which a target can be present. Inparticular, a sample can be a biological sample or a sample obtainedfrom a biological material. Examples of biological samples includetissue samples and cytology samples, with more particular examplesincluding, peripheral blood, urine, saliva, tissue biopsy, surgicalspecimen, amniocentesis samples and autopsy material.

SISH Chromogen A: a silver acetate solution.

SISH Chromogen B: a hydroquinone solution.

SISH Chromogen C: a hydrogen peroxide solution.

Specific Binding Moiety: A member of a specific-binding pair. Specificbinding pairs are pairs of molecules that are characterized in that theybind each other to the substantial exclusion of binding to othermolecules (for example, specific binding pairs can have a bindingconstant that is at least 10³ M⁻¹ greater, 10⁴ M⁻¹ greater or 10⁵ M⁻¹greater than a binding constant for either of the two members of thebinding pair with other molecules in a biological sample). Particularexamples of specific binding moieties include specific binding proteins(for example, antibodies, lectins, avidins such as streptavidins, andprotein A). Specific binding moieties can also include the molecules (orportions thereof) that are specifically bound by such specific bindingproteins.

Target: Any molecule for which the presence, location and/orconcentration is or can be determined. Examples of target moleculesinclude proteins and haptens, such as haptens covalently bonded toproteins. Target molecules are typically detected using one or moreconjugates of a specific binding molecule and a detectable label.

Therapeutically Effective Amount: A quantity of a specified agentsufficient to achieve a desired effect in a subject being treated withthat agent. For example, this may be the amount of a conjugate useful inincreasing resistance to, preventing, ameliorating, and/or treatinginfection and disease. Ideally, a therapeutically effective amount of anagent is an amount sufficient to increase resistance to, prevent,ameliorate, and/or treat infection and without causing a substantialcytotoxic effect in the subject. The effective amount of an agent usefulfor increasing resistance to, preventing, ameliorating, and/or treatinginfection and disease in a subject will be dependent on the subjectbeing treated, the severity of the affliction, and the manner ofadministration of the therapeutic composition.

Vaccine: A vaccine is a pharmaceutical composition that elicits aprophylactic or therapeutic immune response in a subject. In some cases,the immune response is a protective response. Typically, a vaccineelicits an antigen-specific immune response to an antigen of a pathogen,for example, a bacterial or viral pathogen, or to a cellular constituentcorrelated with a pathological condition. A vaccine may include apolynucleotide, a peptide or polypeptide, a polysaccharide, a virus, abacteria, a cell or one or more cellular constituents. In some cases,the virus, bacteria or cell may be inactivated or attenuated to preventor reduce the likelihood of infection, while maintaining theimmunogenicity of the vaccine constituent.

III. Introduction

Certain disclosed embodiments concern a method for forming a conjugateof two or more molecules, and conjugates made by the method. A person ofordinary skill in the art will recognize that the disclosed method isuseful for forming any combination of molecules having functional groupsthat can react with a reactive functional group on a polymeric carrier,such as a hydrazide functional group. The specific conjugates disclosedto exemplify the invention should not be construed to limit the scope ofthe invention. For example, although certain of the disclosed conjugatesare antibody-polymeric hapten carrier conjugates, conjugates betweenother biomolecules and other detectable labels (such as haptens,fluorophores, luminophores, fluorescent labels, fluorescentnanoparticles and fluorescent proteins, such as green fluorescentprotein) also are within the scope of the disclosure.

One embodiment of the disclosed method includes reacting a polymerhaving plural reactive functional groups (polymeric carrier), such asplural reactive hydrazide groups, or combinations of plural differentfunctional groups, with a first molecule (such as an antibody) having agroup that can react with the reactive functional group provided by thepolymeric carrier. For example, if the reactive functional group is ahydrazide, then the functional group can be a carbonyl functional group,such as an aldehyde. The first molecule includes at least one remainingreactive functional group provided by the polymeric carrier that thencan be reacted with a second molecule, such as a hapten directly, ahapten with a linker, or both. Alternatively, a conjugate could beformed comprising a polymeric carrier and a hapten and/or hapten-linker,which is then reacted with the second molecule. In this example, thesecond molecule might be an antibody. As yet another alternative, pluraldifferent polymeric carriers, such as carriers for haptens, may becoupled to a second molecule, such as an antibody. In particularembodiments, the polymeric carrier is a polyacrylic hydrazide that iscoupled to an antibody, preferably solely at the Fc region of theantibody, and plural hapten and/or hapten-linker compounds, such asPEG-biotin, PEG-DNP, fluorescein, etc. are coupled to the polymerichapten carrier.

A further disclosed aspect is a kit that includes a disclosed linker andinstructions for performing the disclosed method for making a conjugate.Also disclosed are methods for using disclosed conjugates to detect atarget in a sample.

IV. Polymeric Carriers

Embodiments of the disclosed invention concern using polymeric materialsas carriers, such as hapten carriers. The polymeric materials consideredgenerally most useful for the present invention have two features: arepeating monomeric unit, or units, characteristic of a particularpolymer; and plural reactive functional groups, where the reactivefunctional groups may be the same or different, that are associated witha repetitive polymeric unit.

A. Polymeric Materials

A person of ordinary skill in the art will appreciate that polymericmaterials other than polyacrylamides can be used to practice disclosedembodiments of the present invention. Solely by way of example, andwithout limitation, these additional polymeric backbone materialsinclude:

1. polyacrylic acids [e.g. (CH₂CHCO₂H)_(n)];

2. polyethyleneimines [e.g. H(NHCH₂CH₂)_(n)NH₂];

3. polystyrenesulfonates, typically having a formula

4. polysaccharides, which are a class of high-molecular-weightcarbohydrates in which monosaccharides are glycosidically joined withthe elimination of water. Polysaccharide typically refers to thosepolymers which contain 10 or more monosaccharide residues.Polysaccharides such as starch, glycogen, dextran, and polyglucosides,may include several thousand units. Polymers of relatively low molecularweight, consisting of two to nine monosaccharide residues, are referredto as oligosaccharides. Unless otherwise noted or the context clearlyindicates otherwise, “polysaccharide” as used herein includes botholigosaccharides and polymers having more than 9 monosaccharideresidues. Polysaccharides, such as cellulose or starch, produce only onemonosaccharide type (D-glucose) on complete hydrolysis, and hence aretermed homopolysaccharides. Heteropolysaccharides, such as hyaluronicacid, produce more than one monosaccharide type on hydrolysis. Withspecific reference to hyaluronic acid the monomers areN-acetylglucosamine and D-glucuronic acid. Exemplary polysaccharidesinclude starch, glycogen, dextran, carboxymethylcellulose, etc.);

5. polyethylene-alt-maleic acid;

6. poly(arginine), poly(aspargine), poly(aspartic acid), poly(glutamicacid), poly(glutamine) and poly(lysine); and

7. polyvinylpyrrolidone (PVP), typically having a formula

A person of ordinary skill in the art will appreciate that derivativesof these exemplary polymeric materials are suitable for use withdisclosed embodiments of the present invention, and further thatpolymeric materials in addition to those disclosed herein to exemplifythe invention also can be used as carriers.

Certain polymeric materials within the disclosure of the presentinvention can be obtained commercially. For example, many of thedisclosed polymers are commercially available from Aldrich in variousmolecular weights. Alternatively, a polymeric material can be purchasedor prepared, and then subsequently derivatized to include desiredfunctional groups. This process can be exemplified by reference to thepolyacrylamide hydrazides, whereby a polyacrylamide is derivatized toinclude plural hydrazide functional groups by microwave mediatedreaction with hydrazine. Similarly, polymers, such as polyacrylamides,may be reacted with any substituted compound, such as aminoguanidines.As yet another alternative approach, monomeric units suitable forforming a desired polymeric backbone, and comprising desired functionalgroups, can be polymerized to form desired polymeric carriers accordingto disclosed embodiments of the present invention. This process allowsusing alternating copolymers comprising regular alternating A and Bunits, periodic copolymers with A and B units arranged in a repeatingsequence (e.g. (A-B-A-B-B-A-A-A-A-B-B-B)_(n), random copolymers withrandom sequences of monomer A and B, statistical copolymers in which theordering of the distinct monomers within the polymer sequence obeysknown statistical rules, block copolymers comprised of two or morehomopolymer subunits linked by covalent bonds, homopolymer units havingan intermediate non-repeating subunit, known as a junction block, blockcopolymers with two or three distinct blocks, such as diblock andtriblock copolymers, linear copolymers having a single main chain,branched copolymers having a single main chain with one or morepolymeric side chains, graft copolymers having side chains structurallydistinct from the main chain, star copolymers, brush copolymers, combcopolymers, dendrimers, etc.

The size of the polymeric material also may be an importantconsideration for certain embodiments of the present application. Forexample, it currently is believed that the average molecular weight ofthe polymeric carrier should be from about 50 to about 100,000, moretypically from about 1,000 to about 50,000, and even more typically fromabout 5,000 to about 40,000, and yet even more typically from about10,000 to about 30,000. Certain disclosed polyacrylamide hydrazideembodiments contemplate using a polymeric material having an averagemolecular weight of 10,000 or less, although polyacrylamides havingsubstantially larger average molecular weights also can be used.Additional guidance for selecting a particular molecular weight ormolecular weight distribution for the polymer can be provided byconsidering physical properties of the polymeric product. For example,the molecular weight of the polymeric carrier can be an importantconsideration, such as for determining the solubility, particularlyaqueous solubility, of the polymeric carrier or the ability of thepolymeric carrier conjugates to penetrate a sample to which a conjugatemay be applied, and hence perform as desired. A person of ordinary skillin the art also will appreciate that an optimal average molecular weightmay well depend on the particular polymeric material, the reactivefunctional group or groups, and the intended use for the material.

Many of the disclosed embodiments are primarily useful for aqueousapplications. As a result, the polymeric carrier preferably should besubstantially soluble in water.

Polymeric backbones used with disclosed embodiments of the presentinvention can be substantially non-crosslinked structures.Alternatively, the polymeric portion can be substantially crosslinked.

B. Reactive Functional Groups

Any reactive functional group that can be used to couple components asdisclosed herein can be useful for practicing the present invention.Certain embodiments concern reactive functional groups where there areat least two adjacent heteroatoms. One purpose for selecting suchfunctional groups is to take advantage of their increasednucleophilicity, such as may result, without limiting the invention to atheory of operation, by the alpha effect, relative to compounds that mayhave a functional group comprising one or more heteroatoms, but nothaving two adjacent heteroatoms. Certain exemplary such functionalgroups are hydrazines, hydrazides, hydroxyl amines (—RNOH), a hydrazidethiol, as disclosed in assignee's prior “Molecular Conjugate” U.S.application Ser. No. 11/603,425.

With reference specifically to hydrazides, such functional groupstypically have a formula —NR—NR₁R₂, where R-R₂ are hydrogen. Thehydrazide may be a substituted hydrazide, i.e. where at least one ofR-R₂ is other than hydrogen, such that R-R₂ independently are hydrogen,aliphatic, such as a lower (typically 20 or fewer, and even moretypically 10 or fewer carbon atoms) alkyl group, heteroaliphatic,aromatic, and/or heteroaromatic. As another example, it also is possibleto use functional groups that are electron donating so that thenucleophilicity of the attacking heteroatom, such as nitrogen, isfurther increased. Suitable functional groups also can be derivatives ofa hydrazide. Exemplary such functional groups include dihydrazides[—(RN)—NR₁CO—NR₂—NR₃R₄], semicarbazides [—NRCO—NR₁NR₂R₃],thiosemicarbazides [—NR—CS—NR1-NR₂R₃], thiocarbazides[—NR—NR₁—CS—NR₂—NR₃R₄], carbonic acid dihydrazine[—NR—CO—NR₁—NR₂—CO—NR₂—NR₄R₅], sulfur containing derivatives of carbonicacid dihydrazines, hydrazine carboxylates [—O—CO—NR—NR₁R₂], or sulfurcontaining derivatives of hydrazine carboxylates, aminoguanidines, etc.With reference to these exemplary groups, R-R₅ typically are hydrogen,but also can be independently hydrogen, aliphatic, heteroaliphatic,aromatic, heteroaromatic, etc.

C. Obtaining Polymeric Carriers

Disclosed embodiments of polymeric carriers generally can be purchasedor can be made using methods known in the art. A number of workingembodiments illustrate polymeric carriers with reference topolyacrylamide hydrazide, where the polymeric backbone is based onacrylamide, and the carrier further comprises plural reactive hydrazidefunctional groups. Polyacrylamide can be made as disclosed in Example 1.

A general formula for polyacrylamide hydrazide is provided below.

With reference to this general formula, X and Y can vary, but typicallyX is from about 100 to about 500, more typically from about 300 to about400, and Y typically is from about 5 to about 100, more typically fromabout 10 to about 50.

Many polymeric hydrazides produced prior to the present inventionapplications were relatively large polymers, and had limited solubility,on the order of about 5 milligram/milliliter or less. Certainembodiments of the present invention are primarily concerned withpolyacrylamide hydrazides having a substantially increased aqueoussolubility of greater than about 5 milligrams/milliliter up to aqueoussaturation, typically greater than about 10 milligrams/milliliter up toat least about 500 milligrams/milliliter, and preferably at least 100milligrams/milliliter, and more preferably at least about 250milligrams/milliliter, and even more preferably at least about 300milligrams/milliliter.

Another important feature is the pKa of the hydrazide functional group,which is around 4. If the pH is about 5 for reactions that may be runusing the polymeric hydrazide, then the hydrazide nitrogen is notprotonated, and therefore capable of acting as a nucleophile. And atthat pH value, the hydrazide acts as a super-nucleophile. For certaindisclosed embodiments, functional groups associated with the polymericcarrier act as nucleophiles to couple with carbonyl compounds producedby oxidizing carbohydrate associated with the Fc portion of an antibody.The nucleophilic hydrazide is a good functional group for this reaction.Conversely, amines have a pKa of greater than 9, and typically greaterthan 10, and hence at a pH of about 5 the amine functional group iscompletely protonated, and hence not nucleophilic. The hydrazide may bereacted with a carbonyl compound, e.g., an aldehyde. The reaction of thehydrazide with the aldehydes can be acid catalyzed. This differenceallows chemoselective reactions. For example, biological amines that maybe present in a sample are fully protonated at a pH of about 5 or less,and hence are not nucleophilic, whereas the hydrazides, hydrazines,hydrazide derivatives, hydrazine derivatives, etc. of the presentinvention are not protonated, and hence available for reactionchemoselectively.

D. Haptens

One of the primary uses of disclosed embodiments is a polymeric carrierfor haptens. Haptens are small molecules that can elicit an immuneresponse, but typically only when coupled to a large carrier, such as aprotein. Any hapten now known or hereafter discovered likely can be usedwith the present invention. Known exemplary haptens includedinitrophenol, biotin, digoxigenin, flourescein, rhodamine,bromodeoxyuridine, and mouse immunoglobulin.

Ventana Medical Systems, Inc. also is the assignee of U.S. patentapplication No. 60/856,133, entitled Haptens, Hapten Conjugates,Compositions Thereof and Method for Their Preparation and Use, which wasfiled Nov. 1, 2006, and corresponding utility application Ser. No.11/982,627, which are incorporated herein by reference. Theseapplications disclose several new classes of haptens, and particularspecies thereof, that are useful for practicing embodiments of thepresent invention. These haptens include pyrazoles, particularlynitropyrazoles; nitrophenyl compounds; benzofurazans; triterpenes; ureasand thioureas, particularly phenyl ureas, and even more particularlyphenyl thioureas; rotenone and rotenone derivatives, also referred toherein as rotenoids; oxazole and thiazoles, particularly oxazole andthiazole sulfonamides; coumarin and coumarin derivatives; cyclolignans,exemplified by Podophyllotoxin and Podophyllotoxin derivatives; andcombinations thereof.

For the general formulas provided below, if no substituent is indicated,a person of ordinary skill in the art will appreciate that thesubstituent is hydrogen. A bond that is not connected to an atom, but isshown, for example, extending to the interior of a ring system,indicates that the position of such substituent is variable. A curvedline drawn through a bond indicates that some additional structure isbonded to that position, typically a linker or the functional group ormoiety used to couple the hapten to a carrier. Moreover, if nostereochemistry is indicated for compounds having one or more chiralcenters, all enantiomers and diasteromers are included. Similarly, for arecitation of aliphatic or alkyl groups, all structural isomers thereofalso are included.

1. Azoles

A first general class of haptens of the present invention is azoles,typically oxazoles and pyrazoles, more typically nitro oxazoles andnitro pyrazoles, having the following general chemical formula.

With reference to this general formula, n is 0-2, most typically 0 or 1.R₁-R₄ can be any organic group that does not interfere with, andpotentially facilitates, the function as a hapten. More specifically,R₁-R₄ independently are selected from: hydrogen, acyl, aldehydes,alkoxy, aliphatic, particularly lower aliphatic, substituted aliphatic,heteroaliphatic, e.g., organic chains having heteroatoms, such asoxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewercarbon atoms, and even more typically lower alkyl having 10 or feweratoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substitutedalkyl, such as alkyl halide (e.g. —CX₃ where X is a halide, andcombinations thereof, either in the chain or bonded thereto,), oxime,oxime ether (e.g., methoxyimine, CH₃—O—N═) alcohols (i.e. aliphatic oralkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, aminoacid, aryl, alkyl aryl, such as benzyl, carbohydrate, monosaccharides,such as glucose and fructose, disaccharides, such as sucrose andlactose, oligosaccharides and polysaccharides, carbonyl, carboxyl,carboxylate (including salts thereof, such as Group I metal or ammoniumion carboxylates), cyclic, cyano (—CN), ester, ether, exomethylene,halogen, heteroaryl, heterocyclic, hydroxyl, hydroxylamine, oxime(HO—N═), keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl,sulfoxide, and combinations thereof. Two or more of these R₁-R₄substituents also may be atoms, typically carbon atoms, in a ring systembonded or fused to the compounds having the illustrated general formula.At least one of the R₁-R₄ substituents is bonded to a linker or is afunctional group suitable for coupling to a linker or a carriermolecule. R₁-R₄ most typically are aliphatic, hydrogen or nitro groups,even more typically alkyl, hydrogen or nitro, and still even moretypically lower (10 or fewer carbon atoms) alkyl, hydrogen, nitro, orcombinations thereof. The number of nitro groups can vary, but mosttypically there are 1 or 2 nitro groups. X independently is nitrogen orcarbon. Y is oxygen, sulfur or nitrogen. If Y is oxygen or sulfur, thenthere is no R₁ group, and n=0. If Y is nitrogen, then there is at leastone R₁ group.

A person of ordinary skill in the art will appreciate that, forcompounds having 2 or more W groups, the relative positions thereof isvariable. For example, a diazole could have nitrogen atoms at the 1 and2 positions, or the 1 and 3 positions. Moreover, more than twoheteroatoms also are possible, such as with triazines.

At least one of R₁-R₄ for these azole compounds is bonded to some othergroup or is a variable functional group. For example, the illustratedcompounds can be coupled either directly to a carrier or to a linker atany of the suitable positions about the azole ring.

Working embodiments typically were mono- or di-nitropyrazolederivatives, such that at least one of R₁-R₄ is a nitro group, andperhaps two of R₁-R₄ are nitro groups, with the remaining R₁-R₄ beingused to couple the hapten to a linker or a carrier.

One particular compound had the following structure.

2. Nitroaryl

A second general class of haptens of the present invention are nitroarylcompounds. Exemplary nitroaryl compounds include, without limitation,nitrophenyl, nitrobiphenyl, nitrotriphenyl, etc., and any and allheteroaryl counterparts, having the following general chemical formula.

With reference to this general formula, such compounds have at leastone, and optionally plural, nitro groups. Thus, at least one of R₁-R₆ isnitro. If more than one of R₁-R₆ is nitro, all combinations of relativering positions of plural nitro substituents, or nitro substituentsrelative to other ring substituents, are included within this class ofdisclosed haptens. Dinitroaryl compounds are most typical. A person ofordinary skill in the art will appreciate that as the number of nitrogroups increases, the number of remaining ring substituents in thegeneral formula decreases. These substituents independently are selectedfrom: hydrogen, acyl, aldehydes, alkoxy, aliphatic, particularly loweraliphatic, substituted aliphatic, heteroaliphatic, e.g., organic chainshaving heteroatoms, such as oxygen, nitrogen, sulfur, alkyl,particularly alkyl having 20 or fewer carbon atoms, and even moretypically lower alkyl having 10 or fewer carbon atoms, such as methyl,ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkylhalide (e.g. —CX₃ where X is a halide, and combinations thereof, eitherin the chain or bonded thereto), oxime, oxime ether (e.g., methoxyimine,CH₃—O—N═) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly loweralkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such asbenzyl, carbohydrate, monosaccharides, such as glucose and fructose,disaccharides, such as sucrose and lactose, oligosaccharides andpolysaccharides, carbonyl, carboxyl, carboxylate (including saltsthereof, such as Group I metal or ammonium ion carboxylates), cyclic,heterocyclic, cyano (—CN), ester, ether, halogen, heteroaryl, hydroxyl,hydroxlyamine, oxime (HO—N═), keto, such as aliphatic ketones, nitro,sulfhydryl, sulfonyl, sulfoxide, exomethylene, and combinations thereof.At least one of the R₁-R₆ substituents is bonded to a linker or is afunctional group suitable for coupling to a linker or a carriermolecule.

Two or more of the R₁-R₆ substituents also may be atoms, typicallycarbon atoms, in a ring system, such as napthalene (shown below) oranthracene type derivatives. Ring systems other than 6-membered ringsystems can be formed, such as fused 6-5 ring systems.

Again, at least on of the ring positions occupied by R₁-R₈ is bonded toa linker or is a variable functional group suitable for coupling, suchas by covalent bonding, to a carrier molecule. For example, nitroarylcompounds of the present invention can include a functional group forcoupling to a carrier, or to a linker, at various optional ringlocations.

Working embodiments are exemplified by nitrophenyl compounds. Solely byway of example, mononitroaryl compounds are exemplified bynitrocinnamide compounds. One embodiment of a nitrocinnamide-basedcompound is exemplified by 4,5-dimethoxy-2-nitrocinnamide, shown below.

The nitrophenyl class of compounds also is represented by dinitrophenylcompounds. At least one of the remaining carbon atoms of the ringpositions not having a nitro group is bonded to a functional group, to alinker, or directly to a carrier. Any and all combinations of relativepositions of these groups are included within the class of disclosedhaptens.

Working embodiments are more particularly exemplified by2,4-dinitrophenyl compounds coupled to a linker, as illustrated below.

R₁-R₃ are as stated above. “L” is a linker, as discussed in more detailbelow.

3. Benzofurazans

Benzofurazans and derivatives thereof are another class of haptenswithin the scope of the present invention. A general formula for thebenzofurazan-type compounds is provided below.

R₁-R₄ substituents independently are selected from: hydrogen, acyl,aldehydes, alkoxy, aliphatic, particularly lower aliphatic, such asisoprene, substituted aliphatic, heteroaliphatic, e.g., organic chainshaving heteroatoms, such as oxygen, nitrogen, sulfur, alkyl,particularly alkyl having 20 or fewer carbon atoms, and even moretypically lower alkyl having 10 or fewer atoms, such as methyl, ethyl,propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide(e.g. —CX₃ where X is a halide, and combinations thereof, either in thechain or bonded thereto,), oxime, oxime ether (e.g., methoxyimine,CH₃—O—N═) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly loweralkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such asbenzyl, carbohydrate, monosaccharides, such as glucose and fructose,disaccharides, such as sucrose and lactose, oligosaccharides andpolysaccharides, carbonyl, carboxyl, carboxylate (including saltsthereof, such as Group I metal or ammonium ion carboxylates), cyclic,heterocyclic, cyano (—CN), ester, alkyl ester, ether, halogen,heteroaryl, hydroxyl, hydroxylamine, oxime (HO—N═), keto, such asaliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene,and combinations thereof. Two or more of these R₁-R₄ substituents alsomay be atoms, typically carbon atoms, in a ring system bonded or fusedto the compounds having the illustrated general formula. At least one ofthe R₁-R₄ substituents is bonded to a linker or directly to a carrier. Yis a carbon atom having R₅ and R₆ substituents, where R₅ and R₆ are asstated for R₁-R₄, oxygen or sulfur, typically oxygen.

Compounds where Y is oxygen are more particularly exemplified bycompounds having the following structure, where R₁-R₄ are as statedabove, and most typically are independently hydrogen and lower alkyl.

One working embodiment of a compound according to this class of haptenshad the following chemical structure.

4. Triterpenes

Triterpenes are another class of haptens within the scope of the presentinvention. The basic ring structure common to the cyclic triterpenes hasfour six-membered fused rings, A-D, as indicated below.

A number of publications discuss naturally occurring, semi-synthetic andsynthetic triterpene species within the genus of triterpenes useful forpracticing the present invention, including: J. C. Connolly and R. A.Hill, Triterpenoids, Nat. Prod. Rep., 19, 494-513 (2002); Baglin et al.,A Review of Natural and Modified Beculinic, Ursolic and EchinocysticAcid Derivatives as Potential Antitumor and Anti-HIV Agents, MiniReviews in Medicinal Chemistry, 3, 525-539; W. N. and M. C. Setzer,Plant-Derived Triterpenoids as Potential Antineoplastic Agents, MiniReviews in Medicinal Chemistry, 3, 540-556 (2003); and Baltina, ChemicalModification of Glycyrrhizic Acid as a Route to New Bioactive Compoundsfor Medicine, Current Medicinal Chemistry, 10, 155-171 92003); each ofwhich is incorporated herein by reference. Based on the presentdisclosure and working embodiments thereof, as well as disclosuresprovided by these prior publications, and with reference to this firstgeneral formula, R₁-R₂₁ independently are selected from: hydrogen, acyl,aldehydes, alkoxy, aliphatic, particularly lower aliphatic, such asisoprene, substituted aliphatic, heteroaliphatic, e.g., organic chainshaving heteroatoms, such as oxygen, nitrogen, sulfur, alkyl,particularly alkyl having 20 or fewer carbon atoms, and even moretypically lower alkyl having 10 or fewer atoms, such as methyl, ethyl,propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide(e.g. —CX₃ where X is a halide, and combinations thereof, either in thechain or bonded thereto,), oxime, oxime ether (e.g., methoxyimine,CH₃—O—N═) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly loweralkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such asbenzyl, carbohydrate, monosaccharides, such as glucose and fructose,disaccharides, such as sucrose and lactose, oligosaccharides andpolysaccharides, carbonyl, carboxyl, carboxylate (including saltsthereof, such as Group I metal or ammonium ion carboxylates), cyclic,heterocyclic, cyano (—CN), ester, alkyl ester, ether, halogen,heteroaryl, hydroxyl, hydroxylamine, oxime (HO—N═), keto, such asaliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene,and combinations thereof. Two or more of these R₁-R₂₁ substituents alsomay be atoms, typically carbon atoms, in a ring system bonded or fusedto the compounds having the illustrated general formula. At least one ofthe R₁-R₂₁ substituents is bonded to a linker or is a functional groupsuitable for coupling to a linker or a carrier molecule. Y is a bond,thereby defining a 5-membered ring, or is a carbon atom bearing R₂₂ andR₂₃ substituents, where these R groups are as stated above.

Disclosed embodiments of triterpenes exemplifying this class of haptensalso may include an E ring, and this E ring can be of various ringsizes, particularly rings having 5-7 atoms, typically carbon atoms, inthe ring. For example, the E ring might be a 6-membered ring, asindicated by the following general formula, where R₁-R₃₁ are as statedabove for R₁-R₂₁.

The following general formulae indicates that the R₁₃ substituent may bean acyl group bearing an R₃₃ substituent selected from hydrogen,hydroxyl, ester, i.e. —OR₃₄ where R₃₄ is aliphatic, typically alkyl orsubstituted alkyl, and even more typically lower alkyl, amido, includingprimary amide (—NH₂), secondary amide (—NHR₃₅) and tertiary amide(—NR₃₅R₃₆), where R₃₅ and R₃₆ are aliphatic, typically lower aliphatic,more typically alkyl, substituted alkyl, and even more typically loweralkyl or substituted lower alkyl. This general formula also indicatesthat the R₁ substituent often is an OR₃₂ substituent, where R₃₂ ishydrogen or aliphatic, more typically alkyl or substituted alkyl, andeven more typically lower alkyl. The remaining R groups are as statedabove with reference to the first general formula.

The E ring also may be a 5 membered ring, as indicated by the formulabelow where the R₁-R₂₉ groups are as stated above for R₁-R₂₁.

With reference to these general formulae, the R₁-R₂₉ groups are asstated above for R₁-R₂₁.

As with exemplary compounds where the E ring is a 6-membered ring,compounds where the E ring is a 5-membered ring also can includesubstituents at R₁ and R₁₃ as discussed above. Specifically, thisgeneral formulae indicates that the R₁₃ substituent may be an acyl groupbearing an R₃₃ substituent selected from hydrogen, hydroxyl, ester, i.e.—OR₃₄ where R₃₄ is aliphatic, typically alkyl or substituted alkyl, andeven more typically lower alkyl, amido, including primary amide (—NH₂),secondary amide (—NHR₃₅) and tertiary amide (—NR₃₅R₃₆), where R₃₅ andR₃₆ are aliphatic, typically lower aliphatic, more typically alkyl,substituted alkyl, and even more typically lower alkyl or substitutedlower alkyl. This general formula also indicates that the R₁ substituentoften is an OR₃₂ substituent, where R₃₂ is hydrogen or aliphatic, moretypically alkyl or substituted alkyl, and even more typically loweralkyl.

Exemplary compounds also include 5-membered rings as both the A and theE ring. General formulae for such exemplary compounds are providedbelow, where the R₁-R₂₉ substituents are as stated above.

Again, the R₁ and R₁₃ substituents can be oxygen-based functionalgroups. The R₁₃ substituent may be an acyl group bearing an R₃₃substituent selected from hydrogen, hydroxyl, ester, i.e. —OR₃₄ whereR₃₄ is aliphatic, typically alkyl or substituted alkyl, and even moretypically lower alkyl, amido, including primary amide (—NH₂), secondaryamide (—NHR₃₅) and tertiary amide (—NR₃₅R₃₆), where R₃₅ and R₃₆ arealiphatic, typically lower aliphatic, more typically alkyl, substitutedalkyl, and even more typically lower alkyl or substituted lower alkyl.This general formula also indicates that the R₁ substituent often is anOR₃₂ substituent, where R₃₂ is hydrogen or aliphatic, more typicallyalkyl or substituted alkyl, and even more typically lower alkyl.

Exemplary triterpenes of the present invention also may include one ormore sites of unsaturation in one or more of the A-E rings. Exemplarycompounds often have at least one site of unsaturation in the C ring,such as the double bond in the C ring as indicated below.

The site of unsaturation may be an alpha, beta unsaturated ketone, suchas illustrated below for the C ring.

The triterpenes also have a number of stereogenic carbon atoms. A personof ordinary skill in the art will appreciate that particular enantiomersare most likely to occur naturally. While the naturally occurringenantiomer may be most available, and/or effective, for practicingdisclosed embodiments, all other possible stereoisomers are within thescope of the present invention. Moreover, other naturally occurringtriterpenes, or synthetic derivatives thereof, or fully syntheticcompounds, may have (1) different stereochemistry, (2) differentsubstituents, and further may be substituted at positions that are notsubstituted in the naturally occurring compounds. The general formulaeprovided above do not indicate stereochemistry at the chiral centers.This is to signify that both enantiomers at each chiral center, and alldiastereomeric isomer combinations thereof, are within the scope of thepresent invention.

Particular working embodiments of the present invention are exemplifiedby the following general formula, in which the substituents are asstated above.

The stereochemistry and substituents for a naturally occurringtriterpene useful as a hapten for practicing the present invention areshown below.

The hydroxyl group in the A ring typically is oxidized to a carbonylfunctional group in working embodiments. As a result, the carbon atombearing the carbonyl group is no longer a chiral center.

5. Ureas and Thioureas

Ureas and thioureas, particularly aryl and heteroaryl ureas andthioureas, are another class of haptens within the scope of the presentinvention. A general formula for urea-based haptens of the presentinvention is provided below.

With reference to this general formula, R₁-R₃ are independentlyhydrogen, aliphatic, substituted aliphatic, typically alkyl, substitutedalkyl, and even more typically lower alkyl and substituted lower alkyl,cyclic, heterocyclic, aryl and heteroaryl. More specifically, R₁typically is aryl or aliphatic, often having at least one site ofunsaturation to facilitate chromophoric detection. R₂ and R₃ mosttypically are independently hydrogen and lower alkyl. Y is oxygen (ureaderivatives) or sulfur (thioureas).

Aryl derivatives typically have the following formula.

R₁-R₇ independently are selected from: hydrogen, acyl, aldehydes,alkoxy, aliphatic, particularly lower aliphatic, such as isoprene,substituted aliphatic, heteroaliphatic, e.g., organic chains havingheteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkylhaving 20 or fewer carbon atoms, and even more typically lower alkylhaving 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, andbutyl, substituted alkyl, such as alkyl halide (e.g. —CX₃ where X is ahalide, and combinations thereof, either in the chain or bondedthereto,), oxime, oxime ether (e.g., methoxyimine, CH₃—O—N═) alcohols(i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl)amido, amino, amino acid, aryl, alkyl aryl, such as benzyl,carbohydrate, monosaccharides, such as glucose and fructose,disaccharides, such as sucrose and lactose, oligosaccharides andpolysaccharides, carbonyl, carboxyl, carboxylate (including saltsthereof, such as Group I metal or ammonium ion carboxylates), cyclic,heterocyclic, cyano (—CN), ester, alkyl ester, ether, halogen,heteroaryl, hydroxyl, hydroxylamine, oxime (HO—N═), keto, such asaliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene,and combinations thereof. At least one of the R₃-R₇ substituents also isbonded to a linker or to a carrier molecule. Two or more of these R₃-R₇substituents available for such bonding also may be atoms, typicallycarbon atoms, in a ring system bonded or fused to the compounds havingthe illustrated general formula.

Additional rings also can be present, as indicated by the exemplarystructures provided below. The R groups are as stated above for R₁-R₇and Y is oxygen or sulfur.

A particular subclass of thioureas is represented below.

With reference to this general formula, n is 1 to 5, typically 1-2, R₁and R₂ are independently hydrogen or lower alkyl, and X independently isa halide or combinations of different halides.

One example of a working embodiment of a phenyl thiourea is providedbelow.

The trifluoromethyl groups are shown in the 3 and 5 positions relativeto the thiourea moiety. A person of ordinary skill in the art willappreciate that compounds having all relative positions fordisubstituted compounds, such as 2,4- and compounds having more than twotrihaloalkyl substituents, at all possible relative positions of suchplural trihaloalkyl substituents, also are within the scope of thepresent invention. A particular example of a rhodamine thiourea haptenhas the following formula.

6. Rotenones

Rotenone and rotenone-based haptens, collectively referred to asrotenoids, provide another class of haptens within the scope of thepresent invention. A first general formula for rotenone, androtenone-based haptens, is provided below.

A number of publications discuss naturally occurring, semi-synthetic andsynthetic rotenoids that are useful for describing the genus ofrotenoids useful for practicing the present invention, including: LeslieCrombie and Donald Whiting, Biosynthesis in the Rotenoids Group ofNatural Products Application of Isotope Methodology, Phytochemistry, 49,1479-1507 (1998); and Nianbai Fang, and John Casida, Cube ResinInsecticide: Identification and Biological Activity of 29 RotenoidConstituents; each of which is incorporated herein by reference. Basedon the present disclosure and working embodiments, as well asdisclosures provided by these prior publications, and with reference tothis first general formula, R₁-R₁₄ independently are hydrogen, aldehyde,alkoxy, aliphatic, particularly lower aliphatic, such as isoprene,substituted aliphatic, heteroaliphatic, e.g., organic chains havingheteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkylhaving 20 or fewer carbon atoms, and even more typically lower alkylhaving 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, andbutyl, substituted alkyl, such as alkyl halide (e.g. —CX₃ where X is ahalide, and combinations thereof, either in the chain or bonded thereto)amino, amino acid, amido, cyano (—CN), halogen, hydroxyl, hydroxylamine,oxime (HO—N═), oxime ether (e.g., methoxyimine, CH₃—O—N═) alkylhydroxyl, particularly lower alkyl hydroxyl, carbonyl, keto, such asaliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, carboxyl,carboxylate (and salts thereof, such as Group I metal or ammonium ioncarboxylates) ester, alkyl ester, acyl, exomethylene, ether, cyclic,heterocyclic, aryl, alkyl aryl, such as benzyl, heteroaryl,polysaccharides, carbohydrate, monosaccharides, such as glucose andfructose, disaccharides, such as sucrose and lactose, oligosaccharidesand polysaccharides, and combinations thereof. Two or more of theseR₁-R₁₄ substituents also may be atoms, typically carbon atoms, in a ringsystem bonded or fused to the compounds having the illustrated generalformula. At least one of the R₁-R₁₄ substituents also is bonded to alinker or to a carrier molecule.

While R₆ and R₇ can be as stated above, such substituents more typicallyindependently are hydrogen, OR₁₅, where R₁₅ is hydrogen, aliphatic,substituted aliphatic, typically alkyl, substituted alkyl, and even moretypically lower alkyl and substituted lower alkyl, such as lower alkylhalides, cyclic, heterocyclic, aryl and heteroaryl, —NR₂₁, where R₂₁ ishydrogen, aliphatic, substituted aliphatic, typically alkyl, substitutedalkyl, and even more typically lower alkyl and substituted lower alkyl,such as lower alkyl halides, cyclic, heterocyclic, aryl and heteroaryl,or N-L-RG, where L is a linker or a reactive group, such as an amine, asdiscussed in more detail herein.

R₆ and R₇ also can form a double bond, such as a double bond to anoxygen to form a carbonyl. If R₆ and/or R₇ are not -L-RG, then at leastone of the R substituents is bonded to a linker or to a carriermolecule.

The B ring also can include at least one additional site ofunsaturation. For example, R₅ and R₁₂ can form a double bond.

R₁₀ and R₁₁ can be joined in a 5- or 6-membered ring. For example, R₁₀and R₁₁ may define a pyran or furan ring, and more particularly is asubstituted and/or unsaturated pyran or furan ring.

Certain exemplary rotenone-based haptens of the present invention alsotypically satisfy the following second general formula.

With reference to this second general formula, the R substituents are asstated above. If R₆ or R₇ is not -L-RG, then at least one of theremaining R groups is bonded to a linker or to a carrier.

R₁₀ and R₁₁ can be joined in a 5- or 6-membered ring, such as a pyran orfuran, and more particularly a substituted and/or unsaturated pyran orfuran ring. Thus, a third general formula useful for describing certainrotenone-based haptens of the present invention is provided below, wherethe R substituents are as stated above.

Y is a bond, thereby defining a 5-membered ring, or is a carbon atom ina 6-membered ring bearing R₁₉ and R₂₀ substituents, as shown below,where the R substituents are as stated above.

R₅ and R₁₂ at the ring juncture are shown without indicating particularstereochemistry. The naturally occurring compound has a cis-ringjuncture, but racemic mixtures also are useful for practicing thepresent invention. Also, the trans stereoisomer quickly equilibrates toform the racemic mixture.

Working embodiments of compounds within this class more typicallysatisfy the following third general formula.

With reference to this general formula, R₆ and R₇ are hydrogen, alkyl,or define a double bond, such as to oxygen to form a carbonyl. R₁₅ andR₁₆ independently are hydrogen and aliphatic, typically lower aliphatic,such as alkenyl, one example of which is isoprene, as shown below.

Again, a particular enantiomer is shown in the above formula, but aperson of ordinary skill in the art will appreciate that the scope ofthe present invention is not limited to the particular enantiomer shown.Instead, all stereoisomers that act as haptens also are within the scopeof the disclosure. All substitutions discussed above for this class ofcompounds applies to this particular compound. Other substitutions alsoare readily apparent to a person of ordinary skill in the art. Forexample, the methoxy groups on the A ring can be any alkoxy compound,particular lower alkoxy groups. The isoprene unit also provides anolefin that can be synthetically modified, perhaps to provide analternative position, or at least a second position, for coupling thehapten to a linker or a carrier molecule. For example, the olefin couldbe converted to an alcohol by hydroboration. It also could be convertedto a halide or an epoxide either for use as a hapten or as intermediatesuseful for further transformation.

A fourth general formula for describing rotenone-based haptens of thepresent invention is particularly directed to rotenone isoxazolines, asprovided below.

R-R₅ independently are hydrogen, aldehyde, alkoxy, aliphatic,particularly lower aliphatic, including all branched chain isomers, suchas isoprene, and all stereoisomers, substituted aliphatic,heteroaliphatic, e.g., organic chains having heteroatoms, such asoxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewercarbon atoms, and even more typically lower alkyl having 10 or feweratoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substitutedalkyl, such as alkyl halide (e.g. —CX₃ where X is a halide, andcombinations thereof, either in the chain or bonded thereto) amino,amino acid, amido, cyano (—CN), halogen, hydroxyl, hydroxylamine, oxime(HO—N═), oxime ether (e.g., methoxyimine, CH₃—O—N═) alkyl hydroxyl,particularly lower alkyl hydroxyl, carbonyl, keto, such as aliphaticketones, nitro, sulfhydryl, sulfonyl, sulfoxide, carboxyl, carboxylate(and salts thereof, such as Group I metal or ammonium ion carboxylates)ester, alkyl ester, acyl, exomethylene, ether, cyclic, heterocyclic,aryl, alkyl aryl, such as benzyl, heteroaryl, polysaccharides,carbohydrate, monosaccharides, such as glucose and fructose,disaccharides, such as sucrose and lactose, oligosaccharides andpolysaccharides, and combinations thereof. At least one of the R-R₅substituents also is bonded to a linker or to a carrier molecule. Y isoxygen, nitrogen, or sulfur.

A particular working embodiment of a rotenone-based hapten satisfyingthis fourth general formula is provided below.

7. Oxazoles and Thiazoles

Oxazole and thiazole sulfonamides provide another class of haptenswithin the scope of the present invention. A general formula for oxazoleand thiazole sulfonamides is provided below.

With reference to this first general formula R₁-R₃ independently areselected from: hydrogen, acyl, aldehydes, alkoxy, aliphatic,particularly lower aliphatic, such as isoprene, substituted aliphatic,heteroaliphatic, e.g., organic chains having heteroatoms, such asoxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewercarbon atoms, and even more typically lower alkyl having 10 or feweratoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substitutedalkyl, such as alkyl halide (e.g. —CX₃ where X is a halide, andcombinations thereof, either in the chain or bonded thereto,), oxime,oxime ether (e.g., methoxyimine, CH₃—O—N═) alcohols (i.e. aliphatic oralkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, aminoacid, aryl, alkyl aryl, such as benzyl, carbohydrate, monosaccharides,such as glucose and fructose, disaccharides, such as sucrose andlactose, oligosaccharides and polysaccharides, carbonyl, carboxyl,carboxylate (including salts thereof, such as Group I metal or ammoniumion carboxylates), cyclic, heterocyclic, cyano (—CN), ester, alkylester, ether, halogen, heteroaryl, hydroxyl, hydroxylamine, oxime(HO—N═), keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl,sulfoxide, exomethylene, and combinations thereof. Two or more of theseR₁-R₃ substituents also may be atoms, typically carbon atoms, in a ringsystem bonded or fused to the compounds having the illustrated generalformula. At least one of the R₁-R₃ substituents is bonded to a linker oris a functional group suitable for coupling to a linker or a carriermolecule. Y is oxygen or sulfur, typically sulfur.

For certain exemplary working embodiments, R₁ has been amido, such asthe amide derivatives shown below. R₂ provides a position for couplingto a linker or to a carrier molecule, although the positions indicatedby R₁ and R₂ also provide alternative or additional positions forcoupling to a linker and/or carrier molecule. R₂, for certain workingembodiments, has been —SO₂, and has been used to couple linkers byforming a sulfonamide. Thus, a second general formula for workingembodiments of haptens exemplifying this class of haptens is indicatedbelow, where the R₃-R₆ substituents and Y are as stated above.

For certain working embodiments R₆ has been alkyl, particularly loweralkyl, such as methyl, and Y has been sulfur.

One working embodiment of a compound according to this class of haptenshad the following chemical structure.

The thiazole or oxazole might also be part of a larger ring system. Forexample, the 5-membered oxazole or thiazole might be coupled to at leastone additional ring, such as a phenyl ring, as indicated below.

While the R₁-R₅ groups generally can be as stated above, such compoundsalso provide a position for coupling to a linker and/or to a carriermolecule, such as a R₅. One possible sulfonamide derivative is providedbelow.

8. Coumarins

Coumarin and coumarin derivatives provide another class of haptenswithin the scope of the present invention. A general formula forcoumarin and coumarin derivatives is provided below.

With reference to this general formula, R₁-R₆ independently are selectedfrom: hydrogen, acyl, aldehydes, alkoxy, aliphatic, particularly loweraliphatic, such as isoprene, substituted aliphatic, heteroaliphatic,e.g., organic chains having heteroatoms, such as oxygen, nitrogen,sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, andeven more typically lower alkyl having 10 or fewer atoms, such asmethyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such asalkyl halide (e.g. —CX₃ where X is a halide, and combinations thereof,either in the chain or bonded thereto,), oxime, oxime ether (e.g.,methoxyimine, CH₃—O—N═) alcohols (i.e. aliphatic or alkyl hydroxyl,particularly lower alkyl hydroxyl) amido, amino, amino acid, aryl, alkylaryl, such as benzyl, carbohydrate, monosaccharides, such as glucose andfructose, disaccharides, such as sucrose and lactose, oligosaccharidesand polysaccharides, carbonyl, carboxyl, carboxylate (including saltsthereof, such as Group I metal or ammonium ion carboxylates), cyclic,heterocyclic, cyano (—CN), ester, alkyl ester, ether, halogen,heteroaryl, hydroxyl, hydroxylamine, oxime (HO—N═), keto, such asaliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene,and combinations thereof. At least one of the R₁-R₆ substituents alsotypically is bonded to a linker or a carrier molecule. Certain workingembodiments have used the position indicated as having an R₅ substituentfor coupling to a linker or carrier molecule. The 4 position can beimportant if fluorescence is used to detect these compounds.Substituents other than hydrogen at the 4 position are believed toquench fluorescence, although such derivatives still may bechromophores. Y is oxygen, nitrogen or sulfur. Two or more of the R₁-R₆substituents available for forming such compounds also may be atoms,typically carbon atoms, in a ring system bonded or fused to thecompounds having the illustrated general formula. Exemplary embodimentsof these types of compounds are provided below.

A person of ordinary skill in the art will appreciate that the ringsalso could be heterocyclic and/or heteroaryl.

Working embodiments typically were fused A-D ring systems having atleast one carrier molecule coupling position, with one possible couplingposition being indicated below.

With reference to this general formula, the R and Y variable groups areas stated above. Most typically, R₁-R₁₄ independently are hydrogen orlower alkyl. Particular embodiments of coumarin-based haptens include2,3,6,7-tetrahydro-11-oxo-1H,5H,11H-[1]benzopyrano[6,7,8-ij]quinolizine-10-carboxylicacid

and diethyl coumarin

9. Cyclolignans

Lignin-based compounds, particularly cyclolignans, such asPodophyllotoxin and derivatives thereof, provide another class ofhaptens within the scope of the present invention. A first generalformula for these cyclolignin-based derivatives is provided below.

A number of publications discuss naturally occurring, semi-synthetic andsynthetic cyclolignans that are useful for describing the genus ofcyclolignans useful for practicing the present invention, including:Stephanie Desbene and Sylviane Giorgi-Renault, Drugs that InhibitTubulin Polymerization The Particular Case of Podophyllotoxin andAnalogues, Curr. Med. Chem.—Anti-Cancer Agents, 2, 71-90 (2002); M.Gordaliza et al., Podophyllotoxin: Distribution, Sources, Applicationsand New Cytotoxic Derivatives, Toxicon, 44, 441-459 (2004); PhillipeMeresse et al., Etoposide: Discovery and Medicinal Chemistry, CurrentMedicinal Chemistry, 11, 2443-2466 (2004); M. Pujol et al., Synthesisand Biological Activity of New Class of Dioxygenated Anticancer Agents,Curr. Med. Chem.—Anti-Cancer Agents, 5, 215-237 (2005); and YoungjaeYou, Podophyllotoxin Derivatives: Current Synthetic Approaches for NewAnticancer Agents, Current Pharmaceutical Design, 11, 1695-1717 (2005);each of which is incorporated herein by reference. Based on the presentdisclosure and working embodiments, as well as disclosures provided bythese prior publications, and with reference to this first generalformula, R₁-R₁₂ typically are selected from hydrogen, aldehyde, alkoxy,aliphatic, particularly lower aliphatic, such as isoprene, substitutedaliphatic, heteroaliphatic, e.g., organic chains having heteroatoms,such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 orfewer carbon atoms, and even more typically lower alkyl having 10 orfewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl,substituted alkyl, such as alkyl halide (e.g. —CX₃ where X is a halide,and combinations thereof, either in the chain or bonded thereto,) amino,amino acid, amido, cyano (—CN), halogen, hydroxyl, hydroxylamine, oxime,oxime ether (e.g., methoxyimine, CH₃—O—N═) alkyl hydroxyl, particularlylower alkyl hydroxyl, carbonyl, keto, such as aliphatic ketones, nitro,sulfhydryl, sulfonyl, sulfoxide, carboxyl, carboxylate (and saltsthereof, such as Group I metal or ammonium ion carboxylates) ester,alkyl ester, acyl, exomethylene, ether, cyclic, heterocyclic, aryl,alkyl aryl, such as benzyl, heteroaryl, polysaccharides, carbohydrate,monosaccharides, such as glucose and fructose, disaccharides, such assucrose and lactose, oligosaccharides and polysaccharides, andcombinations thereof. At least one of R₁-R₁₂ provides a position forcoupling the compound to a linker or to a carrier molecule. Furthermore,certain of the R groups may be atoms in a ring system. For example, R₂and R₃, as well as two of R₇-R₁₀, can be joined together in a ringsystem. At least one of R₁₂ and R₁₁ also often is an aryl group, such asa benzene ring or a substituted benzene ring.

Certain working embodiments also satisfied the following second generalformula, where the R substituents are as stated above.

Exemplary compounds where at least one of R₁₁ and R₁₂ is an aryl grouphave the following general formula, where the R substituents are asstated above.

R₁₆-R₂₀ are generally as stated above, but more typically independentlyare hydrogen or alkoxy, typically lower alkoxy, such as methoxy, asshown below.

At least one of the R substituents typically is bonded to a linker, is areactive functional group capable of reacting with a linker, or is-L-RG. For example, R₅ often is -L-RG.

R₅ and R₆ also may form a double bond, such as a double bond to oxygento form a carbonyl functional group or a double bond to a nitrogen atomto form an imine. Certain exemplary compounds where R₅ and R₆ form adouble bond had the following general formula, where the remaining Rsubstituents are as stated above. Y is selected from nitrogen, oxygen orsulfur. If Y is nitrogen, then the nitrogen atom may further have bondedthereto hydrogen, or some atom, functional group or chemical moietyother than hydrogen. For example, the nitrogen may have an aliphaticsubstituent, such an alkyl group, an aryl or heteroaryl substituent, ora substituted aryl or heteroaryl substituent, such as alkyl and/oralkoxy substituted aryl or heteroaryl substituent.

R₁₆-R₂₀ are independently selected from hydrogen and alkoxy, moretypically lower alkoxy, such as methoxy, as indicated below.

As with all hapten conjugates of the present invention, at least one ofthe R substituents typically is bonded to a linker, is a reactivefunctional group capable of reacting with a linker, is -L-RG, or isdirectly bonded to a carrier. For example, R₉ often is -L-RG.

The chemical structure for Podophyllotoxin, a compound exemplifying thiscyclolignan class of haptens, is provided below.

Podophyllotoxin, also referred to as podofilox, is a non-alkaloid toxinhaving a molecular weight of 414.40 and a compositional formula ofC₂₂H₂₂O₈. Podophyllotoxin is present at concentrations of 0.3 to 1.0% bymass in the rhizome of American Mayapple Podophyllum peltatum. Themelting point of Podophyllotoxin is 183.3-184.0° C.

Accordingly, cyclolignans according to the present invention basedsubstantially on the Podophyllotoxin structure have the followinggeneral formula, where Y is selected from nitrogen, oxygen or sulfur.

A specific example of a cyclolignan hapten according to the presentinvention is shown below.

This compound was made starting with Podophyllotoxin. The hydroxyl groupof Podophyllotoxin was oxidized to a ketone. The ketone was then reactedwith a substituted hydrazine to produce the compound indicated above.The hydrazine reagent can be substituted as desired, including aliphaticand aryl substituents.

10. Heterobiaryl

Another general class of haptens of the present invention isheterobiaryl compounds, typically phenyl quinolines and quinoxalines.Disclosed heterobiaryl compounds have a first general chemical formulaas below.

With reference to this general formulae, A-D are selected from carbon,nitrogen, oxygen, and sulfur, and any and all combinations thereof. Mosttypically A-D are carbon or nitrogen. R₁-R₂ substituents independentlyare selected from: hydrogen, acyl, aldehydes, alkoxy, aliphatic,particularly lower aliphatic, substituted aliphatic, heteroaliphatic,e.g., organic chains having heteroatoms, such as oxygen, nitrogen,sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, andeven more typically lower alkyl having 10 or fewer atoms, such asmethyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such asalkyl halide (e.g. —CX₃ where X is a halide, and combinations thereof,either in the chain or bonded thereto,), oxime, oxime ether (e.g.,methoxyimine, CH₃—O—N═) alcohols (i.e. aliphatic or alkyl hydroxyl,particularly lower alkyl hydroxyl) amido, amino, amino acid, aryl, alkylaryl, such as benzyl, alkoxy aryl, such as methoxy and ethoxy,carbohydrate, monosaccharides, such as glucose and fructose,disaccharides, such as sucrose and lactose, oligosaccharides andpolysaccharides, carbonyl, carboxyl, carboxylate (including saltsthereof, such as Group I metal or ammonium ion carboxylates), cyclic,heterocyclic, cyano (—CN), ester, alkyl ester, ether, halogen,heteroaryl, hydroxyl, hydroxylamine, oxime (HO—N═), keto, such asaliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene,and combinations thereof. Two or more of the R₁-R₂ substituents, mosttypically plural R₁ substituents also may be atoms, typically carbonatoms, in a ring system bonded or fused to the compounds having theillustrated general formula. At least one of the R₁-R₂ substituentstypically is bonded to a linker or directly to a carrier.

Particular embodiments of the heterobiaryl compounds have the followingformula.

R1 and R2 are as stated above for the first general formula. Y isoxygen, nitrogen or sulfur, typically nitrogen. If Y is nitrogen, thenthe formula also can include double bonds to the one or more nitrogenatoms.

Compounds having a single heteroatom are exemplified byphenylquinolines, such as follows.

More particular embodiments include aryl substituted haptens,exemplified by the following general formula.

With reference to this general formula, R₁-R₃ are as indicated above.More typically, R₁ is hydrogen, R₂ is acyl, and R₃ is alkoxy. Aparticular example, 2-(3,4-dimethoxyphenyl)quinoline-4-carboxylic acid,is provided below.

Compounds having two heteroatoms are represented by quinoxalines, asindicated by the general formula below.

A particular example of biaryl-diheteroatom hapten of the presentinvention is exemplified by 3-hydroxy-2-quinoxalinecarbamide, below.Again, the R₁ and R₂ substituents are as stated above with respect tothis class of haptens.

11. Azoaryl

Another general class of haptens of the present invention is azoarylcompounds, such as azobenzenes, having a first general chemical formulaas below.

R₁-R₂ substituents independently are selected from: hydrogen, acyl,aldehydes, alkoxy, aliphatic, particularly lower aliphatic, substitutedaliphatic, heteroaliphatic, e.g., organic chains having heteroatoms,such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 orfewer carbon atoms, and even more typically lower alkyl having 10 orfewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl,substituted alkyl, such as alkyl halide (e.g. —CX₃ where X is a halide,and combinations thereof, either in the chain or bonded thereto,),oxime, oxime ether (e.g., methoxyimine, CH₃—O—N═) alcohols (i.e.aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) amido,amino, amino acid, aryl, alkyl aryl, such as benzyl, alkoxy aryl, suchas methoxy and ethoxy, carbohydrate, monosaccharides, such as glucoseand fructose, disaccharides, such as sucrose and lactose,oligosaccharides and polysaccharides, carbonyl, carboxyl, carboxylate(including salts thereof, such as Group I metal or ammonium ioncarboxylates), cyclic, heterocyclic, cyano (—CN), ester, alkyl ester,ether, halogen, heteroaryl, hydroxyl, hydroxylamine, oxime (HO—N═),keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide,sulfonyl, exomethylene, and combinations thereof. Two or more R₂substituents also may be atoms, typically carbon atoms, in a ring systembonded or fused to the compounds having the illustrated general formula.For example, 2 R₂ substituents may form a fused phenyl ring, or a fusedheterocyclic or heteroaryl structure.

Certain disclosed azoaryl compounds have a first amine substituent and asecond aryl substituent. These compounds typically have the followingformula.

With reference to this general formula, R₂-R₄ are as stated above withrespect to this class of haptens, with particular embodiments havingR₂-R₃ aliphatic, particularly alkyl, more particularly lower alkyl, andR₄ hydrogen.

A third general formula for describing azoaryl compounds is providedbelow.

R₂-R₅ are as stated above for this particular class of haptens. At leastone of R₂-R₅ defines a position for coupling a linker or carrier to theazoaryl hapten to form a conjugate. For example, R₅ may be a sulfonylhalide functional group. Sulfonyl halides, such as that shown below, areuseful functional groups for coupling linkers to the azoaryl haptens.

With reference to this formula, R₂-R₅ are as stated above. X is ahalide. A particular embodiment of these azoaryl haptens,4-(dimethylamino)azobenzene-4′-sulfonyl chloride, has the formulaprovided below.

12. Benzodiazepines

Another class of haptens according to the present invention is thebenzodiazepine haptens, having a first general formula as indicatedbelow.

R₁-R₅ independently are selected from: acyl, aldehydes, alkoxy,aliphatic, particularly lower aliphatic, substituted aliphatic,heteroaliphatic, e.g., organic chains having heteroatoms, such asoxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewercarbon atoms, and even more typically lower alkyl having 10 or feweratoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substitutedalkyl, such as alkyl halide (e.g. —CX₃ where X is a halide, andcombinations thereof, either in the chain or bonded thereto,), oxime,oxime ether (e.g., methoxyimine, CH₃—O—N═) alcohols (i.e. aliphatic oralkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, aminoacid, aryl, alkyl aryl, such as benzyl, carbohydrate, monosaccharides,such as glucose and fructose, disaccharides, such as sucrose andlactose, oligosaccharides and polysaccharides, carbonyl, carboxyl,carboxylate (including salts thereof, such as Group I metal or ammoniumion carboxylates), cyclic, cyano (—CN), ester, ether, exomethylene,halogen, heteroaryl, heterocyclic, hydrogen, hydroxyl, hydroxylamine,oxime (HO—N═), keto, such as aliphatic ketones, nitro, sulfhydryl,sulfonyl, sulfoxide, and combinations thereof. Two or more of the R₅substituents also may be atoms, typically carbon atoms, in a ring systembonded or fused to the compounds having the illustrated general formula.At least one of the R₁-R₅ positions is bonded to a linker or is occupiedby a functional group suitable for coupling to a linker or a carriermolecule. R₁-R₅ most typically are aliphatic, aryl, hydrogen, orhydroxyl, even more typically alkyl, hydrogen or phenyl. Y is oxygen orsulfur, most typically oxygen.

Particular embodiments of the benzodiazepine haptens have R₁ aryl, asindicated below.

For these embodiments, R₂-R₅ are as stated above for this class ofhaptens, more typically such substituents are independently selectedfrom aliphatic, particular alkyl, hydrogen and hydroxyl. Certaindisclosed embodiments are phenyl compounds, as illustrated below.

Again, R₂-R₆ are as stated above, but more typically such substituentsare independently selected from aliphatic, particularly alkyl, hydrogenand hydroxyl. Certain disclosed embodiments are phenyl compounds, asillustrated below. A particular embodiment,4-(2-hydroxyphenyl)-1H-benzo[b][1,4]diazepine-2(3H)-one, is providedbelow.

E. Linkers

-   -   1. General

As indicated by the general formula

hapten-optional linker-carrier

conjugates of the present application may include linkers. Any linkercurrently known for this purpose, or developed in the future, can beused to form conjugates of the present invention by coupling to thehaptens disclosed herein. Useful linkers can either be homo- orheterobifunctional, but more typically are heterobifunctional.

-   -   2. Aliphatic

Solely by way of example, and without limitation, a first class oflinkers suitable for forming disclosed hapten conjugates are aliphaticcompounds, such as aliphatic hydrocarbon chains having one or more sitesof unsaturation, or alkyl chains. The aliphatic chain also typicallyincludes terminal functional groups, including by way of example andwithout limitation, a carbonyl-reactive group, an amine-reactive group,a thiol-reactive group or a photo-reactive group, that facilitatecoupling to haptens and other desired compounds, such as specificbinding moieties. The length of the chain can vary, but typically has anupper practical limit of about 30 carbon atoms. Chain links greater thanabout 30 carbon atoms have proved to be less effective than compoundshaving smaller chain links. Thus, aliphatic chain linkers typically havea chain length of from about 1 carbon atom to about 30 carbon atoms.However, a person of ordinary skill in the art will appreciate that, ifa particular linker has greater than 30 atoms, and still operatesefficiently for linking the hapten to a carrier molecule coupling unit,and the conjugate still functions as desired, then such chain links arestill within the scope of the present invention.

-   -   3. Alkylene Oxides

A second class of linkers useful for practicing the present inventionare the alkylene oxides. The alkylene oxides are represented herein byreference to glycols, such as ethylene glycols. Hapten conjugates of thepresent invention have proved particularly useful if the hydrophilicityof the linker is increased relative to their hydrocarbon chains. As aresult, the alkylene oxides, such as the glycols, have proved useful forpracticing this invention. A person of ordinary skill in the art willappreciate that, as the number of oxygen atoms increases, thehydrophilicity of the compound also may increase. Thus, linkers of thepresent invention generally have a formula of (—OCH₂CH₂O—)_(n) where nis from about 2 to about 25, but more typically n is from about 2 toabout 12.

Heterobifunctional polyalkyleneglycol linkers useful for practicingcertain disclosed embodiments of the present invention are described inassignee's co-pending applications, including “Nanoparticle Conjugates,”U.S. patent application Ser. No. 11/413,778, filed Apr. 28, 2006;“Antibody Conjugates,” U.S. application Ser. No. 11/413,415, filed Apr.27, 2006; and “Molecular Conjugate,” U.S. Provisional Patent ApplicationNo. 60/739,794, filed Nov. 23, 2005; all of which applications areincorporated herein by reference. A person of ordinary skill in the artwill appreciate that the linkers disclosed in these applications can beused to link specific binding moieties, signal generating moieties andhaptens in any and all desired combinations. Heterobifunctionalpolyalkyleneglycol linkers are disclosed below, and their useexemplified by reference to coupling specific binding moieties, such asantibodies, to haptens and detectable labels. In particular, conjugatesof anti-hapten antibodies and detectable labels and conjugates ofprimary antibodies with haptens are exemplified herein.

One particular embodiment of a linker for use with disclosed conjugatesis a heterobifunctional polyalkyleneglycol linker having the generalstructure shown below:

A(CH₂)_(x)—O_(y)B

wherein A and B include different reactive groups, x is an integer from2 to 10 (such as 2, 3 or 4), and y is an integer from 1 to 50, forexample, from 2 to 30 such as from 3 to 20 or from 4 to 12. One or morehydrogen atoms can be substituted for additional functional groups suchas hydroxyl groups, alkoxy groups (such as methoxy and ethoxy), halogenatoms (F, Cl, Br, I), sulfato groups and amino groups (including mono-and di-substituted amino groups such as dialkyl amino groups.

A and B of the linker can independently include a carbonyl-reactivegroup, an amine-reactive group, a hydrazine-reactive group, a hydrazidereactive group, a thiol-reactive group or a photo-reactive group. A andB can be the same group, or can be different groups. Examples ofcarbonyl-reactive groups include aldehyde- and ketone-reactive groupslike hydrazine derivatives and amines. Examples of amine-reactive groupsinclude active esters such as NHS or sulfo-NHS, isothiocyanates,isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals,epoxides, oxiranes, carbonates, aryl halides, imidoesters, anhydridesand the like.

Hapten-linker conjugates have been formed using PEG-based linkers. Oneexample of such a compound is shown below.

This example therefore satisfies the formula hapten-L-RG where L is adPEG₄ (4 ether oxygens) and the reactive group is a carboxylic acidfunctional group. The carboxylic acid functional group has beenconverted to other reactive functional groups in working embodiments.For example, the carboxylic acid functional group can be converted to anactivated ester, such as an NHS ester, as shown below.

And, the activated ester can be converted to other useful reactivefunctional group, such as a hydrazide, as illustrated below.

F. Miscellaneous Materials for Coupling to Polymeric Carriers

Polymeric carriers have plural functional groups through which desiredcompounds or functional groups can be incorporated into conjugates. Forexample, certain disclosed embodiments concern conjugates whereby aportion of available reactive functional groups, such asnitrogen-bearing functional groups having adjacent heteroatoms, forcoupling to a hapten, or haptens, or hapten linkers, leaving a remainingportion of reactive functional groups available for reacting with asecond class of desired molecule. By way of example, and withoutlimitation, the second class of compounds includes: biological molecules(including peptides, proteins, enzymes, sugars, polysaccharides, lipids,glycoproteins, and lipoproteins); detectable labels (linkers having afirst end coupled to the polymeric material and a second coupled to, oravailable for coupling to a desired molecule).

V. Synthesis

A. General

Detailed synthesis of polyacrylamide hydrazide is described in U.S.patent application Ser. No. 11/018,897, which is incorporated herein byreference, and is provided below in Example 1. Briefly, an aqueousmixture of polyacrylamide, commercially available from Sigma Aldrich,and hydrazine monohydrate (Sigma Aldrich) is subjected to microwaveheating. This synthesis is indicated generally below in Scheme 1.

The reaction mixture is purified by precipitation and isolation of thedesired polyacrylamide hydrazide.

Scheme 2 illustrates one embodiment of a microwave mediated synthesis ofpolyvinylpyrrolidone hydrazide.

Scheme 3 illustrates one embodiment of a microwave mediated synthesis ofpolyisobuytlene-co-maleic hydrazide (PIBMH).

Scheme 4 illustrates one embodiment of a synthesis of polyacrylic acidhydrazide. Microwave mediated synthesis of the polyacrylic acidhydrazide polymer leads to little or no product. Without being bound bya theory of operation, it may be that the hydrazine does not react, ordoes not react well, with the free acid functional groups of thepolyacrylic acid, whereas hydrazine does react with amide and acidanhydride functional groups, as illustrated above. As a result, oneembodiment of a successful synthesis is to activate the carboxylic acidfunctional groups, followed by reaction with a protected hydrazine, suchas a BOC-protected hydrazine. A person of ordinary skill in the art willappreciate that acid functional groups may be activated for reactionwith a nucleophile, such as hydrazine or other reactive functionalgroup, in a variety of ways. However, the illustrated working embodimentused 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide (EDAC) to activatethe carboxylic acid functional groups for substitution with aBOC-protected hydrazine. The BOC protecting group was removed withtrifluoroacetic acid (TFA) to produce the polyacrylic acid hydrazidepolymer.

B. Conjugate Synthesis Using Polymeric Hydrazide as an ExemplarySynthesis

While the present invention can be practiced using various polymericcarriers, the following discussion exemplifies the invention withreference to polyacrylamide hydrazide as the polymeric carrier. Thepolyacrylamide hydrazide is coupled to a specific binding molecule, suchas an antibody, as illustrated by FIG. 2. For compounds that include ahydrazide reactive moiety, then no activation of the compound isnecessary. Alternatively and where necessary or desirable, the antibodycan be activated for coupling to a polymeric carrier. For example, oneactivation technique involves providing or producing ahydrazide-reactive functional group on the antibody. A particularlyuseful embodiment of the present invention is coupling the polymericcarrier, such as a polymeric hapten carrier, to the Fc portion of anantibody. To ensure that this reaction occurred, working embodimentstypically have oxidized carbohydrate portions associated with the Fcportion of the antibody to create a hydrazide reactive functional group,typically a compound bearing a carbonyl, such as an aldehyde, orreactive ketone, acid or ester, most typically an aldehyde, using anappropriate oxidizing agent, such as periodate. For working embodiments,excess sodium periodate was used to oxidize proteins.

For the embodiment illustrated in FIG. 2, an intermediate hydrazone isformed by coupling a polymeric carrier to an aldehyde formed at the Fcportion of the antibody. Working embodiments reduced this intermediatehydrazone using an appropriate reagent, such as sodium cyanoborohydride.However, reduction of the intermediate hydrazone may not be required.Working embodiments reduced the intermediate hydrazone to provideincreased stability, such as by eliminating the possibility of aretro-Mannich reaction of the Mannich base. As another example,intermediate hydrazones may be reactive with other components, eitherintra- or inter-molecularly, to produce less desirable compounds, suchas stable heterocycles.

FIG. 2 illustrates that resulting compounds have plural, i.e. “y,”hydrazide functional groups that are available for reaction with anotherdesired compound. This aspect of certain disclosed embodiments isillustrated in FIG. 2, illustrating the principle with particularreference to coupling haptens to the polymeric carrier. As with theantibody, haptens capable of directly reacting with the antibody may notneed to be activated prior to such coupling. Alternatively, a linker maybe used to couple the polymeric carrier-antibody conjugate to a hapten,or haptens, to form a polymeric hapten carrier-antibody conjugate ifactivation is desirable or required, and/or if some other reason favorsusing a linker, such as spacing the hapten from the antibody for stericreasons or to facilitate recognition of the pendent hapten(s).Additional information concerning compositions and uses of linkers canbe found in assignee's patents and/or applications that are incorporatedherein.

FIG. 2 illustrates using various linkers, such as dPEG₄ to dPEG₂₄linkers, and alkyl linkers, such as —C₅H₁₁, to couple DNP, biotin andfluorescein, respectively, to hydrazide functional groups of thepolymeric carrier-antibody conjugate. To facilitate coupling thepolymeric carrier-antibody conjugate to the linker-hapten, the linkerincludes an activated ester, with an N-hydroxysuccinimide (NHS) esterbeing illustrated in FIG. 1. The polymeric carrier-antibody conjugate iscoupled to the hapten to form a polymeric hapten carrier conjugate.

FIG. 2 illustrates that all available “y” groups, that is functionalgroups comprising free hydrazide functional groups, react with theactivated hapten-linker. A person of ordinary skill in the art willappreciate that only a portion of the available reactive functionalgroups may react, such as by using a stoichiometrically limited amountof the linker-hapten. These compounds would have additional reactivefunctional groups available for reacting with, for example, a differenthapten or hapten-linker.

FIG. 2 illustrates first reacting the polymeric carrier with an antibodyto form a polymeric carrier-antibody conjugate, and then coupling ahapten, haptens, hapten-linker, haptens-linker, hapten-linkers, and/orhaptens-linkers (collectively referred to as hapten/hapten-linker), tothe polymeric carrier-antibody conjugate. A person of ordinary skill inthe art will appreciate that the same compounds optionally may be formedby first coupling a polymeric carrier to a hapten/hapten-linker to forma polymeric hapten carrier. The polymeric hapten carrier, havingremaining available reactive functional groups, is then coupled with theantibody, preferably solely at the Fc portion of the antibody, to form apolymeric hapten carrier-antibody conjugate.

VI. Exemplary Disclosed Embodiments of a Method for Using DisclosedPolymeric Hapten Carrier Conjugates, and Compositions Thereof

Certain exemplary embodiments of the present invention concern in situhydridization techniques that can be implemented with variousembodiments of disclosed polymeric hapten carrier conjugates. A samplehaving a target, such as a protein, is selected. A probe useful fordetecting the target, such as an antibody, also is selected. At leastone polymeric hapten carrier is conjugated to the probe. The target istreated with the probe conjugated to the polymeric hapten carrier in amanner effective to form a complex that can be visualized using anysuitable means, such as by treating the target complexed with theprobe-polymeric hapten carrier conjugate with an anti-hapten antibodyhaving a detectable label, such as an enzyme, an organic chromophore,such as a flourphore, chromophoric nanoparticles, such as fluorescentquantum dots, etc., suitable for visualizing the resulting complex. Forexample, if the detectable label is an enzyme, a substrate for theenzyme is provided, thereby producing a uniquely identifiableprecipitate, such as a colored precipitate.

An antibody may be coupled to detectable label, such as an enzyme. Anenzyme substrate is added to produce a detectable enzymatic product. Onespecific embodiment of this process is Silver in situ Hydridization(SISH). One suitable enzyme for SISH is horseradish peroxidase, whichcan be used in combination with hydroquinone, silver ions (e.g., Ag⁺¹)and hydrogen peroxide. The detectable product is elemental silverparticles. Additional information concerning such processes can be foundin Hainfeld, U.S. Pat. No. 6,670,113, which is incorporated herein byreference.

As another example, the enzyme might be alkaline phosphatase. Alkalinephosphatase triggers catalytic hydrolysis of reducing agent phosphate,i.e. ascorbic acid phosphate, generating a reducing agent, i.e.ascorbate, which then may be used to reduce silver plus one (Ag⁺¹) tometallic nanoscopic silver. Thus, the visually detectable product iselemental silver. Silver can be detected by any suitable means,including bright field microscopy. Additional information concerningusing phosphatase enzymes can be found in Bieniarz et al., U.S. PatentApplication No. 2004/0265922, entitled “Enzyme-catalyzed MetalDeposition for the Enhanced in Situ Detection of ImmunohistochemicalEpitopes and Nucleic Acid Sequences,” which is incorporated herein byreference.

Embodiments disclosed herein also can be used to implement ChromogenicIn situ Hydridization. In this process, an enzyme is selected, withsuitable examples including those disclosed herein or that are otherwiseknown to those of ordinary skill in the art, with horseradish peroxidaseand alkaline phosphatase being used to exemplify particular embodiments.A substrate is then selected suitable for producing a coloredprecipitate product that can be detected using techniques known in theart, including bright field microscopy. The chromogenic compound can befluorogenic. Suitable fluorogenic compounds are commercially availablefrom various sources. The substrate can be made fluorogenic by enzymaticaction. Quantum dots also can be used to visualize immunohistochemicalinteractions too. Fluorescent probes and quantum dots typically aremonitored using a fluorescence microscope.

Additional embodiments of a disclosed method concern a direct detectionprocess. For this process, a primary antibody, including a monoclonalantibody, such as mouse monoclonal IgG antibody, is selected for aparticular target. The primary antibody also typically includes adetectable label, as discussed above.

Alternatively, an amplification process can be used. This embodimentalso can be used for diagnostic tests. A target is selected. A primaryantibody is added to the sample in a manner to allow complexation of thetarget and primary antibody. A secondary antibody against the primaryantibody is added to the sample. The antibody includes a detectablelabel that can be used to identify, particularly visually or by visualmeans, such as microscopy, the complexed target using a substrate, asdiscussed herein. The antibody can be any suitable antibody, includingby way of example and without limitation, a labeled rabbit anti-mouseIgG antibody. A secondary antibody, including an antibody from adifferent species, to the primary antibody can be added to the sample.For example, the antibody might be a goat antibody raised against theprimary antibody, such as mouse IgG antibodies.

At least one additional anti-antibody having a detectable label may beadded to the sample to amplify the signal produced by the detectedtarget. In this exemplary process, the antibody might be a labeledrabbit anti-goat IgG antibody. The antibody can be added simultaneouslywith, or subsequent to, as the labeled antibody.

Certain embodiments of the present invention are facilitated by usinganti-hapten monoclonal antibodies, such as for hybridoma screening. Aparticular target is selected, such as a target situated in a tissue. Aprimary antibody directed to the target is administered in a mannereffective for the antibody to recognize the target. The antibody has atleast one, and potentially plural, haptens conjugated thereto usingpolymeric hapten carrier conjugates of the present invention. Thehaptens conjugated to the primary antibody can be the same or different.A tissue sample is treated with anti-hapten antibodies. In thisexemplary embodiment, a primary antibody effectively becomes coupled toan anti-hapten antibody, such as may be provided from a hybridoma mousemonoclonal antibody. Thus, for each hapten coupled to the primaryantibody, there will be a secondary antibody.

The complex formed by the anti-hapten antibody, such as a mousemonoclonal antibody, then is identified. One method is to now treat thecomposition with an antibody that recognizes the mouse antibody, such asa goat antibody. In this exemplary embodiment, goat antibodies areconjugated to a detectable label, such as an enzyme, one example beinghorseradish peroxidase (HRP) enzymes. This complex is then incubatedwith an HRP substrate, as is known to persons of ordinary skill in theart, to form detectable, e.g. colored, precipitates. This process can beused for screening, such as hybridoma screening.

To screen for antihapten monoclonal antibodies, a tissue sample, such asnormal human tonsil tissue, is obtained. The sample may be embedded inparaffin, and if so, the tissue sample is deparaffinized, such as byusing VMSI EZPrep solution. Cell conditioning and antigen retrieval isthen performed using VMSI CC1. A primary polyclonal antibody, such ashuman anti-lambda (available from Dako), is conjugated to embodiments ofpolymeric hapten carriers disclosed in the present application.Conjugation preferably occurs at the Fc region of the antibody to reducethe likelihood that the binding will affect the antibody specificity. Asolution comprising an effective amount of the primary antibody isapplied to the tissue for an effective period of time. For workingembodiments the effective concentration has been about 10 μg/ml of theprimary antibody, and the effective time period has been about 60minutes. The tissue sample is then washed. Thereafter, a potentialanti-hapten antibody (e.g. KLH-CGT1-1.1+5-27F09-02E01) is applied to thetissue sample for an effective period of time, such as about 60 minutes.The antibody is then detected using any suitable means, such as VMSIOmni Map DAB stain.

Automated immunohistochemistry (IHC) screening of potential anti-haptenantibodies can be performed using a VMSI Discovery XT andformalin-fixed, paraffin-embedded human tonsil tissue on glass slides.Tissue samples first undergo deparaffinization, antigen retrieval,followed by the addition of a primary antibody linked to a hapten ofinterest using a polymeric hapten carrier, the potential anti-haptenantibody and a detection antibody. The detection antibody is visualizedusing a chromogen detection reagent from VMSI. Stained slides aremanually screened under a microscope. Samples having a correct primaryantibody staining pattern are selected as potential anti-haptencandidates. To test for selectivity and specificity, candidateanti-hapten cell fusion products are further screened using primaryantibodies conjugated to a hapten of a different chemical class.

Disclosed embodiments contemplate using multiple different haptens, andantibodies thereto, to visualize a detectable target. For example,biotin and DNP haptens, and antibodies thereto, such as antibiotin andanti-DNP, can be used for detection of a target in a sample, such as aprotein in tissue.

Embodiments of the present invention also are useful for simultaneousdetection of multiple different types of targets, such as proteintargets, in a sample. For example, with reference to HER2 (humanepidermal growth factor receptor 2), a polymeric hapten carrier labeledHER2 probe is added to a sample in a manner effective to allow the probeto complex with the HER2 gene. The complexed gene is then treated withan anti-hapten antibody having a detectable label 206, such as a Qdot.An anti-HER2 protein antibody, such as Anti-HER2 4B5 rabbit antibody, isadded to the sample in a manner effective to allow recognition of theHER2 protein. The anti-HER2 antibody may include at least one polymerichapten carrier, and potentially plural haptens, which may be the same ordifferent. With reference to using biotin to exemplify the embodiment,an anti-hapten secondary antibody is then added to the sample in amanner effective to allow complexation of the secondary antibody andhapten(s). Anti-hapten secondary antibody includes a detectable label,such as a Qdot 655. Thus, this embodiment allows multiplexed detectionof gene and gene product.

VII. Test Kits

Disclosed embodiments of the present invention provide, in part, kitsfor carrying out various embodiments of the method of the invention.Examples of such kits include those useful for cholesterol analyses,pregnancy kits, cancer diagnostic kits, etc. Test kits of the presentinvention typically have a polymeric hapten carrier conjugate accordingto the present invention, such as at least one polymeric haptencarrier-specific binding molecule conjugate, including polymeric haptencarrier-antibody conjugates, and an anti-hapten antibody, particularlyan anti-hapten antibody conjugated to a detectable label.

Certain kit embodiments comprise a polymeric hapten carrier-conjugatedantibody, the hapten being selected from oxazoles, pyrazoles, thiazoles,nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones,coumarins, cyclolignans, and combinations thereof. Such kits alsotypically include an anti-hapten antibody conjugated to a detectablelabel.

Further, disclosed kit embodiments can include additional components,including but not limited to plural additional antibodies. Such kits maybe used, for example, by a clinician or physician.

VIII. Automated Embodiments

A person of ordinary skill in the art will appreciate that embodimentsof the method disclosed herein for using hapten conjugates can beautomated. Ventana Medical Systems, Inc. is the assignee of a number ofUnited States patents disclosing systems and methods for performingautomated analyses, including U.S. Pat. Nos. 5,650,327, 5,654,200,6,296,809, 6,352,861, 6,827,901 and 6,943,029, and U.S. publishedapplication Nos. 20030211630 and 20040052685, each of which isincorporated herein by reference. Particular embodiments of polymerichapten staining procedures can be conducted using various automatedprocesses.

Additional details concerning exemplary working embodiments are providedin the working examples.

IX. Examples

The following examples are provided to illustrate certain features ofworking embodiments. A person of ordinary skill in the art willappreciate that the scope of the present invention is not limited to theparticular features exemplified by these examples.

For all disclosed working examples, all chemicals were purchased fromcommercial suppliers and used as received. Solutions of polyclonalantibody (goat anti-mouse and goat anti-rabbit) were purchased fromBethyl Labs and were used as received. Polyacrylamide hydrazide andNHS-PEG₄-DNP were synthesized as previously described. Proteinconcentrations were calculated using ε₂₈₀ values of 1.4 ml mg⁻¹ cm⁻¹ forthe antibody. Water, obtained from an internal deionization source, waspassed through a Milli-Q Biocel System to remove impurities. Bufferexchange was performed using PD-10 columns (GE Biosciences). SEC wasdone using an Akta Purifier (GE Biosciences) and molecular weights arereferenced to protein standards. The flow rate was 1 milliliter/minutethrough a Superdex 200 GL 10/300 column (GE Biosciences).

Example 1

This example describes one embodiment of a method for makingpolyacrylamide hydrazide, as originally disclosed in U.S. patentapplication Ser. No. 11/018,897, which is incorporated herein byreference. Polyacrylamide (1 mmol, 20 mL, 50 wt % solution,Sigma-Aldrich) was mixed with distilled water (10 mL) and hydrazinemonohydrate (20 mL, 420 mmol, Sigma-Aldrich) in a 100 mL round-bottomflask fitted with a condenser. The reaction mixture was microwaved in aCEM Discovery unit for 60 minutes. After cooling to room temperature, anequal volume of methanol was added to the reaction mixture to induceprecipitation. The resulting mixture was centrifuged and decanted. Theresidue was taken up in deionized water (50 mL), and the precipitationprocess repeated for a total of three times. The final residue wasdissolved in deionized water and lyophilized to give a fine, whitehygroscopic powder.

Example 2

This example describes one embodiment of a method for synthesizing anFc-specific haptenylated antibody, as illustrated in FIG. 1. To asolution of polyclonal antibody (1.5 ml, 3.0 mg/ml) was added sodiumperiodate (0.5 ml, 10 mg/ml in deionized water) for a final periodateconcentration of 11.7 mM. The reaction solution was rotated for twohours before desalting with a PD-10 column (0.1 M sodium acetate, 0.15 MNaCl, pH=5.5) to remove unreacted periodate. Thehapten-dPEG_(x)-hydrazide was added in a 500-fold molar excess to theoxidized antibody followed by sodium cyanoborohydride (3.14 mg, 50 μmol)and the reaction was incubated for a period of 18 hours. Size exclusionchromatography (0.1 M Na₃PO₄, 0.15 M NaCl, pH=7.5) gave the purifiedhaptenylated antibody. The number of DNP (ε₃₆₀=18,200 M⁻¹ cm⁻¹;ε₂₈₀=6,500 M⁻¹ cm⁻¹) per antibody was calculated using UV-Vismeasurements while the number of accessible biotin per antibody wasmeasured using an HABA assay available through Sigma-Aldrich.

Example 3

This example describes one embodiment of a method for synthesizing apolyhaptenylated IgG conjugate as illustrated in FIG. 2.

A. Synthesis of Fc-specific PAH Ab

To a solution of polyclonal antibody (1.5 ml, 3.0 mg/ml) was addedsodium periodate (0.5 ml, 10 mg/ml in deionized water) for a finalperiodate concentration of 11.7 mM. The reaction solution was rotatedfor two hours before desalting with a PD-10 column (0.1 M sodiumphosphate, 0.15 M NaCl, pH 7.5) to remove unreacted periodate. Thepolyacrylamide hydrazide linker was added in a 50-fold molar excess tothe antibody along with sodium cyanoborohydride (3.14 mg, 50 μmol) andthe reaction was incubated for a period of 18 hours. SEC (0.1 M sodiumacetate, pH 5.0) yielded the purified antibody-PAH conjugate.

B. Synthesis of Polyhaptenylated Antibody

To a solution of the PAH-IgG (2.0 ml, 0.53 mg/ml) was addedNHS-dPEG_(x)-hapten (50-fold excess) and the reaction was incubated fora period of 18 hours. SEC (0.1 M sodium phosphate, 0.15 M NaCl, pH=7.5)resulted in the purified polyhaptenylated antibody. The number ofhaptens per antibody was calculated using UV-Vis measurements while thenumber of accessible biotin-per-antibody was measured using an HABAassay available through Sigma-Aldrich.

Example 4

This example describes one embodiment of a method for synthesizing achemoselective Fc-specific polyacrylamide hydrazide-antibody conjugateas illustrated generally in FIG. 2. A solution of a polyclonal antibody(0.8 m/L of 1.0 mg/mL) was incubated with a 100 mM aqueous solution ofsodium periodate (0.2 ml) for two hours at room temperature. Thesolution was buffer exchanged by passing through a column of G-25 (GELifesciences, PD-10 column) using ABS (0.10 M sodium acetate, 0.15MNaCl, pH 5.5). PAH was added to the oxidized Ab using a 50-fold molarexcess to the Ab and incubated at room temperature for one hour. Sodiumcyanoborohydride (50 molar excess) was added and incubated for 18 hoursat room temperature. The PAH-Ab was purified on a size exclusion columnusing ABS (0.10 M acetate, 0.15M NaCl, pH 5.5). The NHS-dPEG_(x)-hapten(10-100× molar excess) was added and the reaction was incubated for aperiod of 18 hours. SEC (0.1 M sodium phosphate, 0.15 M NaCl, pH=7.5)resulted in the purified polyhaptenylated antibody. The number ofhaptens per antibody was calculated using UV-Vis measurements while thenumber of accessible biotin-per-antibody was measured using an HABAassay available through Sigma-Aldrich. The number of haptens was lessthan conjugates in Example 3, but higher than Example 2.

Example 5

This example describes one embodiment of a method for synthesizing anitropyrazole-labeled polyacrylamide hydrazide-antibody conjugate asillustrated generally in FIG. 2.

Purified polyacrylamide hydrazide-antibody conjugate in ABS (0.10Msodium acetate, 0.15 M NaCl, pH 5.5) was incubated with a 20-fold molarexcess of nitropyrazole-dPEG₈-NHS for 18 hours. The mixture was purifiedby size-exclusion chromatography using PBS (0.10 M sodium phosphate,0.15 M NaCl, pH 7.2) to yield poly-nitropyrazole-PAH-Ab. The number ofnitropyrazoles per PAH-Ab was determined by UV-Vis measurements.

Example 6

This example describes one embodiment of a method for synthesizing abenzofurazan-labeled polyacrylamide hydrazide-antibody conjugate asillustrated generally in FIG. 2. Purified polyacrylamidehydrazide-antibody conjugate in ABS (0.10 M sodium acetate, 0.15 M NaCl,pH 5.5) was incubated with 20-fold molar excess ofbenzofurazan-dPEG₈-NHS for 18 hours. The mixture was purified bysize-exclusion chromatography using PBS (0.10 M sodium phosphate, 0.15 MNaCl, pH 7.2) to yield poly-benzofurazan-PAH-Ab. The number ofbenzofurazans per PAH-Ab was determined by UV-Vis measurements.

Example 7

This example describes one embodiment of a method for synthesizingdinitrophenyl-labeled polyacrylamide hydrazide-antibody conjugate asillustrated generally in FIG. 2. Purified polyacrylamidehydrazide-antibody conjugate in ABS (0.10 M sodium acetate, 0.15 M NaCl,pH 5.5) was incubated with a 100-fold molar excess ofdinitrophenyl-dPEG₈-NHS for 18 hours. The mixture was purified bysize-exclusion chromatography using PBS (0.10 M sodium phosphate, 0.15 MNaCl, pH 7.2) to yield poly-dinitrophenyl-PAH-Ab. The number ofdinitrophenyls per PAH-Ab was determined by UV-Vis measurements.

Example 8

This example describes one embodiment of a method for synthesizingthiazolesulfonamide-labeled polyacrylamide hydrazide-antibody conjugateas illustrated generally in FIG. 2. Purified polyacrylamidehydrazide-antibody conjugate in ABS (0.10 M sodium acetate, 0.15 M NaCl,pH 5.5) was incubated with a 20-fold molar excess ofthiazolesulfonamide-dPEG₈-NHS for 18 hours. The mixture was purified bysize-exclusion chromatography using PBS (0.10 M sodium phosphate, 0.15 MNaCl, pH 7.2) to yield poly-thiazolesulfonamide-PAH-Ab. The number ofthiazolesulfonamides per PAH-Ab was determined by UV-Vis measurements.

Example 9

This example concerns detecting tissue epitopes, particularly Ki-67 ontonsil, using quantum dots to recognize a secondary antibody conjugatedwith a polyhaptenylated polymer. The following is the adapted procedurefrom the Ventana Benchmark Instrument. The paraffin coated tissue on theslide was heated to 75° C. for 4 minutes and treated twice with EZPrepvolume adjust (VMSI) at 75° C. before application of the liquid coverslip (VMSI) with EZPrep volume adjust. After 4 minutes at 75° C., theslide was rinsed and EZPrep volume adjust was added along with liquidcover slip to deparaffin the tissue at 76° C. for 4 minutes. The slidewas cooled to 40° C. and rinsed three times before the addition of amouse anti-Ki67 (100 μL, VMSI) antibody followed by liquid cover slipand incubation at 40° C. for 16 minutes. After rinsing the slide, thetissue was treated with a goat anti-mouse-PAH-biotinylated antibody (100μL) followed by liquid cover slip and incubation at 40° C. for 8minutes. The slide was rinsed twice with buffer followed by theapplication of liquid cover slip and the addition of 655 nm QDot-SAconjugate (100 μL, 20 mmol) and incubation at 37° C. for 16 minutes. Theslide was rinsed three times with buffer and treated to a detergent washbefore manual application of a cover slip to the slide, after which theslide was viewed through a microscope. FIGS. 3-6 illustrate stainingresults obtained according to this example, with FIGS. 5 and 6illustrating staining results obtained using a 10-fold dilution of theprimary antibody.

Example 10

This example illustrates the evaluation of anti-lambda on tonsil usingquantum dots conjugated directly to secondary anti-hapten antibodies asillustrated generally in FIG. 7. The procedure is an adaptation of theautomated staining protocol from the Ventana Benchmark Instrument. Theparaffin coated tissue on the slide was heated to 75° C. for 8 minutesand treated twice with EZPrep, volume adjusted (VMSI) at 75° C. beforeapplication of the liquid cover slip (VMSI). After two 8 minuteincubation times at 75° C., the slide was rinsed and EZPrep volumeadjusted, followed with liquid coverslip to deparaffinize the tissue.The slide was cooled to 37° C., incubated for 2 minutes and rinsed oncewith reaction buffer. The slide was then treated with cell conditionertwice, followed by liquid coverslip. The slide is heated to 95° C. for 8minutes, followed by coverslip, then is heated to 100° C. for 4 minutes,followed by coverslip. “Apply cell conditioner, incubate for 4 minutes,apply coverslip”, this incubation process with cell conditioner wasrepeated 9 times at 100° C. Slide was cooled down for 8 minutes, rinsedwith reaction buffer, volume adjust, followed by liquid coverslip. Theslide is heated to 37° C. for 2 minutes and rinsed two times before theaddition of the primary conjugate (anti-Lambda-PAH-dPEG₈-hapten, 100 μL,VMSI) followed by liquid cover slip and incubation at 37° C. for 32minutes. The slide was rinsed twice with reaction buffer followed by theapplication of liquid cover slip and the addition of the appropriateanti-hapten Ab-quantum dot conjugate (100 μL, 20-50 nmol) and incubatedat 37° C. for 32 minutes. The slide was rinsed two times with bufferfollowed by liquid coverslip. The slide is removed from the instrumentand treated with a detergent wash before manual application of a coverslip. The slide image was captured by using a CRI Imaging camera on afluorescent microscope with a long-pass filter and image enhancementsoftware (Acquity). FIGS. 8-11 illustrate staining results obtainedaccording to this example.

Example 11

This example concerns evaluating alkaline phosphatase-antibodymultimeric conjugates, particularly HPV in different tissues usingFc-hydrazide-dPEG_(x)-hapten conjugates followed by AP-IgG detection asdepicted in FIG. 12. The following is an adapted procedure from theVentana Benchmark Instrument: the paraffin coated tissue on the slidewas heated to 75° C. for 4 minutes and treated twice with EZPrep volumeadjust (VMSI) at 75° C. before application of the liquid cover slip(VMSI) with EZPrep volume adjust. After 4 minutes at 75° C., the slidewas rinsed and EZPrep volume adjust was added along with liquid coverslip to deparaffin the tissue at 76° C. for 4 minutes. Cell Conditioner#2 (VMSI) was added, the slide warmed to 90° C. and incubated for 8minutes. This was followed by another application of Cell Conditioner #2and incubation at 90° C. for 12 minutes. The slide was rinsed withReaction Buffer (VMSI), cooled to 37° C. and ISH-Protease 3 (100 μL,VMSI) was added. After an incubation of 4 minutes, the slide was rinsedthree times before the application of iView+HybReady (200 μL, VMSI)which was incubated for 4 minutes. Addition of HPV HR Probe (200 μLVMSI) was followed by an incubation of 4 minutes at 37° C., 12 minutesat 95° C. and 124 minutes at 52° C. The slide was then rinsed twice andwarmed to 72° C. This last step was repeated two more times beforecooling the slide down to 37° C. and adding iView+Anti-DNP (100 μL,VMSI). The primary antibody was incubated for 20 minutes and the slidewas then rinsed twice before the manual addition of the polyacrylamidehydrazide biotinylated secondary (goat anti-rabbit, 100 μL, 10 μg/ml).Incubation of the secondary occurred for 20 minutes and the slide twice.The anti-hapten antibody was then applied (100 μL) and incubationoccurred for another 20 minutes. After two more rinse steps, the goatanti-rabbit AP conjugate was applied (100 μL, 6 μg/ml) and incubated for8 minutes. Four more rinse steps were followed by the application of theiView+Enhancer (100 μL, VMSI) which was followed by incubation for 4minutes and application of both iView+NBT (100 μL, VMSI) and iView+BCIP(100 μL, VMSI). The slide was then incubated for a period of 24 minutes,rinsed three times, and Counterstain NFR (100 μL, VMSI) was added. Afterincubation with the counterstain for 4 minutes, the slide was rinsedthree more times and taken off of the instrument. The slide was treatedto a detergent wash before dehydration with ethanol, acetone and xyleneand subsequent application of a cover slip to the slide, after which theslide was viewed through a microscope.

Example 12

This example concerns evaluating horseradish peroxidase-antibodymultimeric conjugates, particularly evaluation of HPV in differenttissues using Fc-conjugated biotin-hydrazide or biotinylatedpolyacrylamide hydrazide for SISH detection as depicted in FIG. 13. Thefollowing is an adapted procedure from the Ventana Benchmark Instrument:the paraffin coated tissue on the slide was heated to 75° C. for 4minutes and treated twice with EZPrep volume adjust (VMS 1) at 75° C.before application of the liquid cover slip (VMSI) with EZPrep volumeadjust. After 4 minutes at 75° C., the slide was rinsed and EZPrepvolume adjust was added along with liquid cover slip to deparaffin thetissue at 76° C. for 4 minutes. Cell Conditioner #2 (VMSI) was added,the slide warmed to 90° C., and incubated for 8 minutes. This wasfollowed by another application of Cell Conditioner #2 and incubation at90° C. for 12 minutes. The slide was rinsed with Reaction Buffer (VMSI),cooled to 37° C. and ISH-Protease 3 (100 μL, VMSI) was added. After anincubation of 4 minutes, the slide was rinsed three times before theapplication of iView+HybReady (100 μL, VMSI) which was incubated for 4minutes. Addition of HPV HR Probe (200 μL VMSI) was followed by anincubation of 4 minutes at 37° C., 12 minutes at 95° C. and 124 minutesat 52° C. The slide was then rinsed twice and warmed to 72° C. This laststep was repeated two more times before cooling the slide down to 37° C.and adding iView+Anti-DNP (100 μL, VMSI). The primary antibody wasincubated for 20 minutes and the slide was then rinsed twice before themanual addition of the polyacrylamide hydrazide-biotinylated secondary(goat anti-rabbit, 100 μL, 10 μg/ml). Incubation of the secondaryoccurred for 8 minutes and the slide twice. The rabbit anti-biotinantibody was then applied (100 μL) and incubation occurred for another20 minutes. After two more rinse steps, the HRP multimer was applied(100 μL, 10 μg/ml) and incubated for 8 minutes. Four more rinse stepswere followed by the application of the SISH Chromagen A (100 μL VMSI)with a 4 minute incubation, SISH Chromagen B (100 μL, VMSI) with a 4minute incubation, and SISH Chromagen C (100 μL, VMSI) with a 4 minuteincubation. The slide was rinsed three times, and Hematoxylin II (100μL, VMSI) was added. After incubation with the counterstain for 4minutes, the slide was rinsed and Bluing Reagent (100 μL, VMSI) wasapplied and incubated for 4 minutes. The slide was then rinsed threemore times and taken off of the instrument. The slide was treated to adetergent wash before dehydration with ethanol, acetone and xylene andsubsequent application of a cover slip to the slide, after which theslide was viewed through a microscope. FIGS. 14-21 illustrate stainingresults obtained according to this example.

Example 13

This example illustrates the multiplexed detection of anti-Kappa, CD34,CD45 and Ki-67 on tonsil with quantum dots as depicted generally in FIG.22. The procedure is an adaptation the automated staining protocol fromthe Ventana Benchmark Instrument. The paraffin coated tissue on theslide was heated to 75° C. for 8 minutes and treated twice with EZPrep(VMSI), volume adjusted at 75° C. before application of the liquid coverslip (VMSI). After two 8 minute incubation times at 75° C., the slidewas rinsed and EZPrep volume adjusted, followed with liquid coverslip todeparaffinize the tissue. The slide was cooled to 37° C., incubated for2 minutes and rinsed once with reaction buffer. The slide was thentreated with cell conditioner twice, followed by liquid coverslip. Theslide is heated to 95° C. for 8 minutes, followed by coverslip, then isheated to 100° C. for 4 minutes, followed by coverslip. “Apply cellconditioner, incubate for 4 minutes, apply coverslip”, this incubationprocess with cell conditioner was repeated 9 times at 100° C. Slide wascooled down for 8 minutes, rinsed with reaction buffer, volume adjust,followed by liquid coverslip. The slide is heated to 37° C. for 2minutes and rinsed two times before the addition of the primaryconjugates (anti-Kappa-PAH-dPEG₈-dinitrophenyl,—CD34-PAH-dPEG₈-nitropyrazole, —CD45-PAH-dPEG₈-thiosulfonamide andKi-67-PAH-dPEG₈-benzofuran, 100 μL of each, VMSI) followed by liquidcover slip and incubation at 37° C. for 32 minutes. The slide was rinsedtwice with reaction buffer and the appropriate cocktail of anti-haptenAb-Quantum Dot conjugates (100 μL each, 20-50 nmol) followed by theapplication of liquid cover slip and incubated at 37° C. for 32 minutes.The slide was rinsed two times with buffer followed by liquid coverslip.The slide is removed from the instrument and treated with a detergentwash before manual application of a cover slip. The slide image wascaptured by using a CRI Imaging camera on a fluorescent microscope witha long-pass filter and image enhancement software (Acquity). FIGS. 23-26illustrate staining results obtained according to this example.

Example 14

This example concerns the synthesis of dextran hydrazide, dextranhydrazines, dextran amines, and dextran guanidines. DextranAldehydes(Pierce) average MW 10,000, 20,000 or 40,000 containing between 10 to200 aldehydes are dissolved in phosphate buffer pH 7.0.

Corresponding bis-PEG_(x)-amine, bis-hydrazide PEG_(x)-hydrazide,bis-PEG_(x)-hydrazine or guanidine-containing linker e.g. aminoguanidine(Aldrich), are added as a buffered pH 7.0 solution in very large molarexcess (100× with the aldehyde content the limiting reagent on thedextran carrier).

The reaction is stirred at room temperature for 1-3 hours. A largeexcess (300× of the reducing agent e.g. sodium cyanoborohydride,Aldrich) is added as an aqueous solution from an addition funnel, over aperiod of 2 hours.

Reaction solutions are stirred overnight and then dialyzed against waterseveral times, utilizing an appropriate dialysis tubing which will allowdiffusion of the smaller MW molecules and retaining the largederivatized carrier.

The dialyzed solutions of the carriers are lyophilized and stored at2-8° C. as desiccated powders. The number of amines, hydrazines,guanidines or hydrazides may be quantitated according to the methodsdescribed in the literature.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

Example 15

This example describes one embodiment of a method for makingpolyvinylpyrrolidone hydrazide (PVPH). Polyvinylpyrrolidone (1 mmol, 20mL, 50 wt % solution, Sigma-Aldrich) was mixed with hydrazinemonohydrate (50 mL, 1.0 mol, Sigma-Aldrich) in a 100 mL round-bottomflask fitted with a condenser. The reaction mixture was microwaved in aCEM Discovery unit for 60 minutes at 120° C. at various powers (100 W,200 W, 300 W). The reaction was reduced in vacuo to an off-white foam.The residue was taken up in a minimal amount of DI water and mixed witha large volume of tetrahydrofuran (THF) to induce precipitation. Theresulting mixture was centrifuged and decanted. The residue was taken upin a minimal amount of deionized water, and the precipitation processwith THF repeated for a total of three times. The final residue wasdissolved in deionized water and lyophilized to give a fine, off-whitehygroscopic powder.

Example 16

This example describes one embodiment of a method for makingpolyisobuytlene-co-maleic hydrazide (PIBMH). Polyisobutylene-co-maleicanhydride (7.1 mmol, 1.09 g, Sigma-Aldrich) was mixed with hydrazinemonohydrate (7.0 mL, 144 mmol, Sigma-Aldrich) in a 10 mL CEM microwavetube. The reaction mixture was microwaved in a CEM Discovery unit for 60minutes at 120° C. at 300 W. The reaction was reduced in vacuo to anoff-white foam. The residue was taken up in a minimal amount of DI waterand mixed with a large volume of ethanol to induce precipitation. Theresulting mixture was centrifuged and decanted. The residue was taken upin a minimal amount of deionized water, and the precipitation processwith ethanol repeated for a total of three times. The final residue wasdissolved in deionized water and lyophilized to give a fine, off-whitehygroscopic powder.

Example 17

This example describes one embodiment of a method for making polyacrylicacid hydrazide (PAAH). Polyacrylic acid (9.57 mmol, 2.00 g, 45% wtsolution in water, Sigma-Aldrich) was diluted with 40 mL DI water andreacted with t-butyl carbazate (9.57 mmol, 1.24 g, Sigma-Aldrich) andEDAC (19.1 mmol, 3.66 g) at room temperature for 14 hours. The reactionmixture pH was adjusted to <3 by dropwise addition of 1M HCl to induceprecipitation of the polymer. The polymer precipitate was filtered,washed with DI water and vacuum dried to produce 846 mg of material. TheBOC-protected polymer was stirred in trifluoroacetic acid (8 mL) untilcompletely dissolved over one hour and the TFA removed in vacuo. Theresidual TFA was removed by azeotropic distillation with toluene,followed by methylene chloride, and further dried under reduced pressureto give 720 mg of a white solid.

Example 18

This example describes the method for determining the molar equivalentsof reactive hydrazide groups per polymer by fluorescence. A standardcurve was generated by reacting six concentrations (0.1 to 0.8 mM) ofacetylhydrazide with fluorescamine in PBS, pH 7.5 and plotting thefluorescence (A360/IE460) vs. concentration. The R² value was 0.994 forthe standard curve.

Polyacrylamide, polyisobutylene-co-maleic anhydride,polyvinylpyrrolidone, and polyacrylic acid were used as negativecontrols for hydrazide incorporation in polyacrylamide hydrazide,polyvinylpyrrolidone hydrazide, polyisobutylene-co-maleic hydrazide andpolyacrylic acid hydrazide as synthesized from Examples 1, 15, 16 and17, respectively. Each polymer was dissolved in PBS, pH 7.5 to a knownconcentration, reacted with fluorescamine, and the fluorescence measuredat 460 nm. The number of reactive hydrazides was calculated compared tothe standard curve and adjusted for the average molecular weight of theindividual polymers.

Hydrazide incorporation was highest for polyacrylamide, butpolyisobutylene-co-maleic anhydride and polyvinylpyrrolidone producedfunctionalized polymer. Using additional microwave energy did not appearto significantly increase hydrazide functionalization as demonstrated inTable 2. The non-microwave-mediated incorporation of hydrazine inpolyacrylic acid produced higher hydrazide incorporation than eitherpolyisobutylene-co-maleic anhydride or polyvinylpyrrolidone, but lesserincorporation than polyacrylamide.

TABLE 1 Polymer Hydrazide Incorporation Molar Equivalents Polymer ofHydrazides Polyacrylamide 0.0 Polyacrylamide hydrazide 76Polyisobutylene-co-maleic anhydride 0.0 Polyisobutylene-co-maleichydrazide 38 Polyvinylpyrrolidone 4.7 Polyvinylpyrrolidone hydrazide 30Polyacrylic acid 0.0 Polyacrylic acid hydrazide 53

TABLE 2 Polymer Hydrazide Incorporation by Microwave Power MolarEquivalents Polymer of Hydrazides Polyvinylpyrrolidone 4.7Polyvinylpyrrolidone hydrazide (100 W) 33 Polyvinylpyrrolidone hydrazide(200 W) 34 Polyvinylpyrrolidone hydrazide (300 W) 30

Example 19

This example describes one embodiment of a method for synthesizing achemoselective Fc-specific polyvinylpyrrolidone hydrazide-antibody(PVPH-Ab) conjugate as illustrated generally in FIG. 2. A solution of apolyclonal antibody (0.8 mL of 1.0 mg/mL) was incubated with a 100 mMaqueous solution of sodium periodate (0.2 ml) for two hours at roomtemperature. The solution was buffer exchanged by passing through acolumn of G-25 (GE Lifesciences, PD-10 column) using ABS (0.10 Macetate, 0.15M NaCl, pH 5.5). PVPH was added to the oxidized Ab using a50-fold molar excess to the Ab and incubated at room temperatureovernight. The PVPH-Ab was purified on a size exclusion column using ABS(0.10 M acetate, 0.15M NaCl, pH 5.5). The NHS-dPEG₈-DNP (50× molarexcess) was added and the reaction was incubated for a period of 18hours. SEC (0.1 M phosphate, 0.15 M NaCl, pH=7.5) resulted in thepurified polyhaptenylated antibody. The number of haptens per antibodywas calculated using UV-Vis measurements.

Example 20

This example describes one embodiment of a method for synthesizing achemoselective Fc-specific polyisobutylene-co-maleic hydrazide-antibody(PIBM-Ab) conjugate as illustrated generally in FIG. 2. A solution of apolyclonal antibody (0.8 mL of 1.0 mg/mL) was incubated with a 100 mMaqueous solution of sodium periodate (0.2 ml) for two hours at roomtemperature. The solution was buffer exchanged by passing through acolumn of G-25 (GE Lifesciences, PD-10 column) using ABS (0.10 Macetate, 0.15M NaCl, pH 5.5). PVPH was added to the oxidized Ab using a50-fold molar excess to the Ab and incubated at room temperatureovernight. The PIBMH-Ab was purified on a size exclusion column usingABS (0.10 M acetate, 0.15M NaCl, pH 5.5). The NHS-dPEG₈-DNP (50× molarexcess) was added and the reaction was incubated for a period of 18hours. SEC (0.1 M phosphate, 0.15 M NaCl, pH=7.5) resulted in thepurified polyhaptenylated antibody. The number of haptens per antibodywas calculated using UV-Vis measurements.

Example 21

This example describes one embodiment of a method for synthesizing achemoselective Fc-specific polyacrylic acid hydrazide-antibody (PAAH-Ab)conjugate as illustrated generally in FIG. 2. A solution of a polyclonalantibody (0.8 mL of 1.0 mg/mL) was incubated with a 100 mM aqueoussolution of sodium periodate (0.2 ml) for two hours at room temperature.The solution was buffer exchanged by passing through a column of G-25(GE Lifesciences, PD-10 column) using ABS (0.10 M acetate, 0.15M NaCl,pH 5.5). PAAH was added to the oxidized Ab using a 50-fold molar excessto the Ab and incubated at room temperature overnight. The PAAH-Ab waspurified on a size exclusion column using ABS (0.10 M acetate, 0.15MNaCl, pH 5.5). The NHS-dPEG₈-DNP (50× molar excess) was added and thereaction was incubated for a period of 18 hours. SEC (0.1 M phosphate,0.15 M NaCl, pH=7.5) resulted in the purified polyhaptenylated antibody.The number of haptens per antibody was calculated using UV-Vismeasurements.

Example 22

This example concerns detecting tissue epitopes, particularly Ki-67 ontonsil, using either chromogenic staining (i.e. HRP-mediated depositionof DAB) or quantum dots to recognize an antibody conjugated with apolyhaptenylated polymer. The following is the adapted procedure fromthe Ventana Benchmark Instrument. The paraffin-coated tissue on theslide was heated to 75° C. for 4 minutes and treated twice with EZPrepvolume adjust (VMSI) at 75° C. before application of the liquid coverslip (VMSI) with EZPrep volume adjust. After 4 minutes at 75° C., theslide was rinsed and EZPrep volume adjust was added along with liquidcover slip to deparaffin the tissue at 76° C. for 4 minutes. The slidewas cooled to 40° C. and rinsed three times before the addition of amouse anti-Ki67 (100 μL, VMSI) antibody followed by liquid cover slipand incubation at 40° C. for 16 minutes. After rinsing the slide, thetissue was treated with a goat anti-mouse-PVPH-DNP antibody (100 μL)followed by liquid cover slip and incubation at 40° C. for 8 minutes.The slide was rinsed twice with buffer followed by the application ofliquid cover slip and the addition of 655 nm QDot:anti-DNP MAb conjugate(100 μL, 20 nmol) and incubation at 37° C. for 16 minutes. The slide wasrinsed three times with buffer and treated to a detergent wash beforemanual application of a cover slip to the slide, after which the slidewas viewed through a microscope. FIGS. 27 and 28 illustrate stainingresults obtained according to this example.

Example 23

This example concerns detecting tissue epitopes, particularly Ki-67 ontonsil, using either chromogenic staining (i.e. HRP-mediated depositionof DAB) or quantum dots to recognize an antibody conjugated with apolyhaptenylated polymer. The following is the adapted procedure fromthe Ventana Benchmark Instrument. The paraffin-coated tissue on theslide was heated to 75° C. for 4 minutes and treated twice with EZPrepvolume adjust (VMSI) at 75° C. before application of the liquid coverslip (VMSI) with EZPrep volume adjust. After 4 minutes at 75° C., theslide was rinsed and EZPrep volume adjust was added along with liquidcover slip to deparaffin the tissue at 76° C. for 4 minutes. The slidewas cooled to 40° C. and rinsed three times before the addition of amouse anti-Ki67 (100 μL, VMSI) antibody followed by liquid cover slipand incubation at 40° C. for 16 minutes. After rinsing the slide, thetissue was treated with a goat anti-mouse-PIBMH-DNP antibody (100 μL)followed by liquid cover slip and incubation at 40° C. for 8 minutes.The slide was rinsed twice with buffer followed by the application ofliquid cover slip and the addition of 655 nm QDot:anti-DNP MAb conjugate(100 μL, 20 nmol) and incubation at 37° C. for 16 minutes. The slide wasrinsed three times with buffer and treated to a detergent wash beforemanual application of a cover slip to the slide, after which the slidewas viewed through a microscope. FIGS. 29 and 30 illustrate stainingresults obtained according to this example.

Example 24

This example concerns detecting tissue epitopes, particularly Ki-67 ontonsil, using either chromogenic staining (i.e. HRP-mediated depositionof DAB) or quantum dots to recognize an antibody conjugated with apolyhaptenylated polymer. The following is the adapted procedure fromthe Ventana Benchmark Instrument. The paraffin-coated tissue on theslide was heated to 75° C. for 4 minutes and treated twice with EZPrepvolume adjust (VMSI) at 75° C. before application of the liquid coverslip (VMSI) with EZPrep volume adjust. After 4 minutes at 75° C., theslide was rinsed and EZPrep volume adjust was added along with liquidcover slip to deparaffin the tissue at 76° C. for 4 minutes. The slidewas cooled to 40° C. and rinsed three times before the addition of amouse anti-Ki67 (100 μL, VMSI) antibody followed by liquid cover slipand incubation at 40° C. for 16 minutes. After rinsing the slide, thetissue was treated with a goat anti-mouse-PAAH-DNP antibody (100 μL)followed by liquid cover slip and incubation at 40° C. for 8 minutes.The slide was rinsed twice with buffer followed by the application ofliquid cover slip and the addition of 655 nm QDot:anti-DNP MAb conjugate(100 μL, 20 nmol) and incubation at 37° C. for 16 minutes. The slide wasrinsed three times with buffer and treated to a detergent wash beforemanual application of a cover slip to the slide, after which the slidewas viewed through a microscope. FIGS. 31 and 32 illustrate stainingresults obtained according to this example.

The present application has been described with reference to certainparticular embodiments. A person of ordinary skill in the art willappreciate that the scope of the invention is not limited to thoseparticular embodiments.

1. A method for forming a molecular conjugate, comprising coupling aspecific binding molecule to a detectable label through a reactivefunctional group provided by a polymeric carrier, the reactivefunctional group being selected from hydrazines, hydrazides, hydrazinederivatives, hydrazide derivatives, guanidines, aminoguanidines,hydroxyl amines, or combinations thereof.
 2. The method according toclaim 1 where the polymeric carrier comprises a polymeric portionselected from polyacrylamide, polyacrylamide-N-hydroxysuccinimides,polyacrylic acids, polyethyleneimines, polysaccharides,polyethylene-alt-maleic acids, polyamino acids or polyvinylpyrrolidones.3. The method according to claim 1 where the polymeric carrier ispolyacrylamide hydrazide or polyvinylpyrrolidone hydrazide.
 4. Themethod according to claim 1 where the polymeric carrier is apolyacrylamide hydrazide having an average molecular weight of 10,000 orless.
 5. The method according to claim 3 where the polyacrylamidehydrazide is non-thiolated.
 6. The method according to claim 1 where thespecific binding molecule is Avidin, an antibody, a nucleic acid, apeptide nucleic acid (PNA), or an aptamer.
 7. The method according toclaim 1 where the specific binding molecule is an antibody.
 8. Themethod according to claim 1, comprising: forming a first compound bycoupling the specific binding molecule to at least a portion of thereactive functional groups; and coupling at least a portion of remainingreactive functional groups of the first compound to the detectablelabel.
 9. The method according to claim 1, comprising: forming a firstcompound by coupling the detectable label to at least a portion of thereactive functional groups; and coupling at least a portion of remainingreactive functional groups of the first compound to the specific bindingmolecule.
 10. The method according to claim 7 comprising coupling thepolymeric carrier to an Fc portion of the antibody through reactivehydrazide functional groups.
 11. The method according to claim 10further comprising activating the antibody for reaction with thereactive functional groups.
 12. The method according to claim 11comprising chemically modifying a glycosylated portion of the antibody.13. The method according to claim 1 where the detectable label is anenzyme, a fluorophore, a luminophore, a hapten, a fluorescentnanoparticle, or combinations thereof.
 14. The method according to claim13 where the hapten is selected from di-nitrophenyl, biotin,digoxigenin, fluorescein, rhodamine, bromodeoxyuridine, mouseimmunoglobulin, or combinations thereof.
 15. The method according toclaim 13 where the hapten is an oxazole, a pyrazole, a thiazole, abenzofurazan, a triterpene, a urea, a thiourea, a nitroaryl other thandinitrophenyl, a rotenoid, a coumarin, a cyclolignan, a heterobiaryl, anazoaryl, or a benzodiazepine, or combinations thereof.
 16. The methodaccording to claim 13 where the hapten is coupled to the specificbinding molecule through the reactive functional groups using an NHS-PEGlinker.
 17. The method according to claim 1 where the detectable labelis a hapten.
 18. The method according to claim 17 where plural differenthaptens are coupled to the polymeric carrier.
 19. The method accordingto claim 1 further comprising reacting a third molecule with at leastone of the reactive functional groups.
 20. The method according to claim1, comprising: oxidizing an Fc portion of an antibody to form anoxidized Fc portion; coupling the polymeric carrier to the oxidized Fcportion of the antibody through at least one reactive hydrazidefunctional group to form a first compound; and coupling the firstcompound to at least one hapten through a reactive hydrazide functionalgroup to form a second compound.
 21. The method according to claim 20where the hapten is coupled to the specific binding molecule throughreactive hydrazide functional groups using an NHS-PEG linker.
 22. Themethod according to claim 20 comprising coupling the first compound toplural haptens.
 23. The method according to claim 20 comprising couplingthe first compound to plural different haptens.
 24. The method accordingto claim 2 where the polysaccharide is selected from carbohydrates,cellulose, carboxymethylcellulose, dextran, amido dextrans, hydrazidedextrans, hydrazine dextrans, glycogen, polyhyaluronic acid and starch.25. The conjugate according to claim 2 where the polyamino acid isselected from poly(aspargine), poly(aspartic acid), poly(glutamic acid),poly(glutamine), poly(lysine), poly(guanidines), or combinationsthereof.
 26. The method according to claim 1 where the molecularconjugate is a polyhaptenylated conjugate.
 27. A conjugate producedaccording to the method of claim
 1. 28. A conjugate, comprising aspecific binding molecule covalently bonded to a detectable labelthrough a reactive functional group provided by a polymeric linkercomprising at least one reactive functional group selected fromhydrazines, hydrazides, hydrazine derivatives, hydrazide derivatives,guanidines, aminoguanidines, hydroxyl amines, or combinations thereof.29. The conjugate according to claim 28 where the polymeric linkercomprises a polymeric portion selected from polyacrylamide,polyacrylamide-N-hydroxysuccinimides, polyacrylic acids,polyethyleneimines, polysaccharides, polyethylene-alt-maleic acids,polyamino acids or polyvinylpyrrolidones, the polymeric portionincluding plural reactive functional groups selected from hydrazines,hydrazides, hydrazine derivatives, hydrazide derivatives, guanidines,aminoguanidines, hydroxyl amines, or combinations thereof.
 30. Theconjugate according to claim 28 where the polymeric linker comprises apolyacrylamide or polyvinylpyrrolidone portion.
 31. The conjugateaccording to claim 28 where the specific binding molecule is Avidin, anantibody, a nucleic acid, a peptide nucleic acid (PNA), or an aptamer.32. The conjugate according to claim 28 where the specific bindingmolecule is an antibody.
 33. The conjugate according to claim 28 wherethe polymeric linker is a PEG-based hydrazide linker.
 34. The conjugateaccording to claim 28 where the detectable label is an enzyme, afluorophore, a luminophore, a hapten, a fluorescent nanoparticle, orcombinations thereof.
 35. The conjugate according to claim 28 where thedetectable label is a hapten selected from di-nitrophenyl, biotin,digoxigenin, fluorescein, rhodamine, bromodeoxyuridine, mouseimmunoglobulin, or combinations thereof.
 36. The conjugate according toclaim 28 where the detectable label is a hapten selected from oxazoles,pyrazoles, thiazoles, benzofurazans, triterpenes, ureas, thioureas,nitroaryls other than dinitrophenyl, rotenoids, coumarins, cyclolignans,heterobiaryls, azoaryls, benzodiazepines, or combinations thereof. 37.The conjugate according to claim 28 where the polymeric linker is apolyacrylamide hydrazide, having plural hydrazide functional groups,that is coupled to an oxidized Fc portion of an antibody and to at leastone detectable label through hydrazide groups.
 38. The conjugateaccording to claim 28 comprising a polyhaptenylated conjugate.
 39. Theconjugate according to claim 29 where the polysaccharide is selectedfrom carbohydrates, cellulose, carboxymethylcellulose, dextran, amidodextrans, hydrazide dextrans, hydrazine dextrans, glycogen,polyhyaluronic acid and starch.
 40. The conjugate according to claim 29where the polyamino acid is selected from poly(aspargine), poly(asparticacid), poly(glutamic acid), poly(glutamine), poly(lysine),poly(guanidines), or combinations thereof.
 41. The method according toclaim 28 where the molecular conjugate is a polyhaptenylated conjugate.42. A method for performing a diagnostic assay for a target in a sample,comprising: contacting the sample with a specific binding molecule thatbinds specifically to a target, wherein the specific binding molecule isconjugated to a detectable label through a polymeric linker comprisingplural reactive functional groups selected from hydrazines, hydrazides,hydrazine derivatives, hydrazide derivatives, guanidines,aminoguanidines, hydroxyl amines, or combinations thereof; and detectingthe specific binding molecule bound to the target using the detectablelabel.
 43. The method according to claim 42 where the polymeric linkerincludes a polymeric portion selected from polyacrylamide,polyacrylamide-N-hydroxysuccinimides, polyacrylic acids,polyethyleneimines, polysaccharides, polyethylene-alt-maleic acids,polyamino acids or polyvinylpyrrolidones.
 44. The method according toclaim 42 where the polymeric linker is a polyacrylamide hydrazide orpolyvinylpyrrolidone hydrazide.
 45. The method according to claim 42where the specific binding molecule is Avidin, an antibody, a nucleicacid, a peptide nucleic acid (PNA), or an aptamer.
 46. The methodaccording to claim 42 where the specific binding molecule is anantibody.
 47. The method according to claim 42 where the detectablelabel is an enzyme, a fluorophore, a luminophore, a hapten, afluorescent nanoparticle, or combinations thereof.
 48. The methodaccording to claim 50 where the hapten is selected from di-nitrophenyl,biotin, digoxigenin, fluorescein, rhodamine, bromodeoxyuridine, mouseimmunoglobulin, or combinations thereof.
 49. The method according toclaim 50 where the hapten is selected from oxazoles, pyrazoles,thiazoles, benzofurazans, triterpenes, ureas, thioureas, nitroarylsother than dinitrophenyl, rotenoids, coumarins, cyclolignans,heterobiaryls, azoaryls, benzodiazepines or combinations thereof. 50.The method according to claim 42 where the detectable label is a haptenand the method further comprises contacting the sample with ananti-hapten antibody.
 51. The method according to claim 53 furthercomprising contacting the sample with an anti-antibody antibody.
 52. Themethod according to claim 42, where the specific binding molecule isconjugated to a detectable label by a nonthiolated, polyacrylamidehydrazide carrier having a molecular weight of 10,000 or less andcomprising plural reactive hydrazide functional groups.
 53. The methodaccording to claim 42 where the assay is a multiplexed diagnostic assayfor two or more different targets in a sample, the method comprising:contacting the sample with two or more specific binding molecules thatbind specifically to two or more different targets, where the two ormore specific binding molecules are conjugated to different haptensthrough reactive functional group of the polymeric linker; andcontacting the sample with two or more different anti-hapten antibodiesthat can be detected separately.
 54. The method according to claim 43where the polysaccharide is selected from carbohydrates, cellulose,carboxymethylcellulose, dextran, amido dextrans, hydrazide dextrans,hydrazine dextrans, glycogen, polyhyaluronic acid and starch.
 55. Themethod according to claim 43 where the polyamino acids are selected frompoly(arginine), poly(aspargine), poly(aspartic acid), poly(glutamicacid), poly(glutamine) and poly(lysine), or combinations thereof. 56.The method according to claim 42 where the specific binding molecule isan antibody comprising an oxidixed Fc portion and the polymeric linkeris coupled to the oxidized Fc portion through a reactive functionalgroup.
 57. The method according to claim 47 where the hapten is coupledto the specific binding molecule through a reactive functional group ofthe polymeric carrier using an NHS-PEG linker.
 58. The method accordingto claim 42 where plural different haptens are coupled to the polymericlinker.
 59. The method according to claim 42 where the specific bindingmolecules are antibodies, and the method comprises: contacting thesample with two or more primary antibodies, each of the primaryantibodies being conjugated to different haptens; and contacting thesample with two or more secondary anti-hapten antibodies, each of thesecondary anti-hapten antibodies being conjugated to different quantumdots.